Adenoviruses and methods for using adenoviruses

ABSTRACT

This invention relates to methods and materials for nucleic acid delivery, vaccination, and/or treating cancer. More specifically, methods and materials for nucleic acid delivery, vaccination, and/or treating cancer using one or more recombinant adenoviruses (Ads) as an oncolytic agent are provided.

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 112 KB file named“ADZE_1_SEQUENCE_LISTING_2023_FILED” created on 12 Jul. 2023.

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINTINVENTOR QUALIFYING FOR THE GRACE PERIOD PROVIDED UNDER 35 USC §102(b)(1)

The applicant submits that the following disclosures qualify under thegrace period provided as noted above: Nguyen, et al., OncolyticVirotherapy 8:43-51, May 3, 2018; Nguyen, et al., Virology 514:118-123,15 Jan. 2018; Matchett, et al., J Virol., 93:1-18, May 1, 2019.

BACKGROUND OF THE INVENTION Technical Field of the Invention

This invention relates to methods and materials for nucleic aciddelivery, vaccination, and/or treating cancer. For example, theinvention encompasses adenoviruses (Ads) and methods for usingadenoviruses to treat medical conditions such as cancer. In an aspect ofthe invention, an adenovirus provided herein can be used as an oncolyticagent.

Despite vast efforts, cancer remains a major public health issue in theUnited States with over 1.6 million new cases in 2017 alone (NationalCancer Institute, “Cancer Stat Facts: Cancer of Any Site,”seer.cancer.gov/statfacts/html/all.html). Traditional therapies, such aschemotherapeutics, radiation therapy and surgery, often fail, especiallywhen cancer is advanced. One of the reasons is for this that cancercells can eliminate or modify the components that are targeted by thesetherapies and effectively avoid being killed.

Oncolytic virotherapy can provide an alternative approach to cancertreatment by utilizing selectively replicating viruses to destroytumors, activate adaptive immune responses, and ensure a life-longimmunity against the tumors (Russell et al., 2017 Molecular Therapy25:1107-1116).

This invention provides methods and materials for nucleic acid delivery,vaccination, and/or treating cancer. For example, this inventionprovides methods and materials for treating cancer by administering oneor more recombinant Ads (e.g., one or more of Ad657 and variantsthereof) as an oncolytic agent. In an embodiment, a recombinant Ad canbe derived from a first Ad (e.g., can include a genome of a first Ad,such as Ad6, also referred to as the recombinant Ad backbone) and caninclude hexon HVRs from a second Ad such as Ad57. In cases where arecombinant Ad includes an Ad6 genome and Ad57 hexon HVRs, therecombinant Ad can be a chimeric Ad referred to as Ad657. (See Nguyen,et al. Oncolytic Virotherapy 7:43-51, 2018, the disclosure of which isincorporated by reference).

In an aspect, this invention provides methods for vaccinating againstinfectious disease using one or more recombinant Ads (e.g., one or moreof Ad657 and variants thereof). In an aspect, this invention providesmethods for treating cancer using one or more recombinant Ads (e.g., oneor more of Ad657 and variants thereof) as an oncolytic agent. In somecases, one or more recombinant Ads (e.g., one or more Ad657s) can beused to reduce the number of cancer cells (e.g., by infecting andkilling cancer cells) in a mammal. In some cases, one or morerecombinant Ads (e.g., one or more of Ad657 and variants thereof) can beused to stimulate anti-cancer immune responses in a mammal. In somecases, one or more recombinant Ads (e.g., one or more of Ad657 andvariants thereof) can be used to stimulate immune responses againstinfectious diseases in a mammal.

As demonstrated herein, when Ad657 is delivered by intravenous injectionto mice having subcutaneous human DU145 prostate cancer tumors, Ad657first infects the liver and then reaches distant tumors. Both Ad6 andAd657 mediated significant delays in tumor growth and extension ofsurvival with Ad6 mediating higher efficacy.

This invention provides methods and materials for nucleic acid delivery,vaccination, and/or treating cancer. For example, this inventionprovides methods and materials for treating cancer by administering oneor more recombinant Ads (e.g., one or more of Ad657 and variantsthereof) as an oncolytic agent. In an embodiment, a recombinant Ad canbe derived from a first Ad (e.g., can include a genome of a first Ad,such as Ad6, also referred to as the recombinant Ad backbone) and caninclude hexon HVRs from a second Ad such as Ad57. In cases where arecombinant Ad includes an Ad6 genome and Ad57 hexon HVRs, therecombinant Ad can be a chimeric Ad referred to as Ad657. In an aspect,this invention provides methods for vaccinating against infectiousdisease using one or more recombinant Ads (e.g., one or more of Ad657and variants thereof). In an aspect, this invention provides methods fortreating cancer using one or more recombinant Ads (e.g., one or more ofAd657 and variants thereof) as an oncolytic agent. In some cases, one ormore recombinant Ads (e.g., one or more Ad657s) can be used to reducethe number of cancer cells (e.g., by infecting and killing cancer cells)in a mammal. In some cases, one or more recombinant Ads (e.g., one ormore of Ad657 and variants thereof) can be used to stimulate anti-cancerimmune responses in a mammal. In some cases, one or more recombinant Ads(e.g., one or more of Ad657 and variants thereof) can be used tostimulate immune responses against infectious diseases in a mammal.

As demonstrated herein, when Ad657 is delivered by intravenous injectionto mice having subcutaneous human DU145 prostate cancer tumors, Ad657first infects the liver and then reaches distant tumors. Both Ad6 andAd657 mediated significant delays in tumor growth and extension ofsurvival with Ad6 mediating higher efficacy.

Moreover, liver sequestration is a considerable problem for virtuallyany oncolytic virus if it is used as an intravenous systemic therapy. Ifthe virus infects hepatocytes and kills them, this will result in liverdamage at low doses and death at higher doses. Notably, administrationof the Ads of the invention, i.e., Ad657 chimeric vector and variantsthereof, mediated unexpected lower liver damage than either Ad5 or Ad6.Thus the unique combination of Ad6 platform with the HVRs 1-7 of Ad657mediated changes in biodistribution and therapy not observed in naturalviruses.

Also, as demonstrated herein, immunization of rhesus macaques withreplicating single-cycle adenovirus (SC-Ad657) vaccines expressing onlyclade B HIV-1 gp160 by intranasal (IN) and intramuscular (IM) routes wascompared to mucosal and systemic routes of vaccination. SC-Ad vaccinesby themselves generated significant circulating antibody titers againstEnv after only a single immunization. Animals immunized only by the IMroute had high peripheral T follicular helper (pTfh) cells in blood, butlow Tfh in lymph nodes, and had lower antibody-dependent cellularcytotoxicity (ADCC) antibody activity. Animals immunized by the IN routehad high Tfh in lymph nodes, but low pTfh in the blood, and had higherADCC antibodies. When immunized animals were challenged rectally withSHIV_(SF162P3), they all became infected, but mucosally-primed animalshad markedly lower viral loads their gastrointestinal tracts. Similarly,Ad657 carrying genes for hepatitis C antigens is able to generatecytotoxic T lymphocyte (CTL) responses against hepatitis andcytomegalovirus. Ad657 is able to delivery and express therapeutic genesincluding cytokines like 4-1BBL, granulocyte macrophage stimulatingfactor (GMCSF), and IL-21. The results provided herein demonstrate thatrecombinant Ads can be used as a local or systemic delivery vehicle fornucleic acid, vaccines, and/or oncolytic virotherapy for cancers.

BRIEF SUMMARY OF THE INVENTION

In general, one aspect of this invention features recombinant Adcomprising (a) an Ad genome from a first Ad strain and (b) a nucleicacid encoding a hexon polypeptide from a second Ad strain, where one ormore of the hypervariable regions the hypervariable regions (HVRs) ofthe hexon polypeptide are different from the HVRs encoded by the Adgenome. The first Ad strain can be a first human Ad strain, and thesecond Ad strain can be a second human Ad strain which is different fromthe first human Ad strain. The first Ad strain and the second Ad straincan be serotypically distinct. The first Ad strain can be a human Ad6strain, and the second Ad strain can be a human Ad57 strain. Therecombinant Ad also can include one or more targeting polypeptides,antigenic polypeptides, enzymes, amino acid substitutions, PEGylation,ligands, tags and the like.

A recombinant Ad may be used as a vector for gene-based vaccination, forgene therapy application/delivery, or for oncolytic virotherapy.

In a further embodiment, the recombinant Ad comprises (a) an Ad genomefrom a first Ad strain and (b) a nucleic acid encoding at least onehexon polypeptide from one or more Ad strains, where the hypervariableregions (HVRs) of the hexon polypeptide from the one or more Ad strainsare different from the HVRs encoded by the first Ad genome.

In a further embodiment, the recombinant Ad can be a replicationcompetent or conditionally-replicating Ad (e.g., a CRAd).

In another aspect, this invention features a recombinant and/or chimericAd comprising (a) nucleic acid encoding a first hexon polypeptide and(b) a second hexon polypeptide, where the amino acid sequence of thefirst hexon polypeptide is different from the amino acid sequence of thesecond hexon polypeptide. The amino acid sequence of a hypervariableregion (HVR) of the first hexon polypeptide can be different from theamino acid sequence of a hypervariable region of the second hexonpolypeptide. The nucleic acid can be from a first Ad strain, and thesecond hexon polypeptide can be from a second Ad strain. The first Adstrain can be a first human Ad strain, and the second Ad strain can be asecond human Ad strain different from the first human Ad strain. Thefirst Ad strain and the second Ad strain can be serotypically distinct.The Ad strain can be a human Ad6 strain, and the second Ad strain can bea human Ad57 strain. The recombinant Ad also can include a targetingpolypeptide. The targeting polypeptide can include the amino acidsequence TARGEHKEEELI (SEQ ID NO:1).

In a further embodiment, the recombinant Ad comprises a) nucleic acidencoding a first hexon polypeptide and (b) a second hexon polypeptidefrom one or more Ad strains, where the amino acid sequence of the firsthexon polypeptide is different from the amino acid sequence of thesecond hexon polypeptide from the one or more Ad strains.

In a further embodiment, the recombinant Ad can be a replicationcompetent Ad or conditionally-replicating Ad (e.g., a CRAd).

In another aspect, the invention provides materials and methods fortreating a mammal having cancer. The methods can include, or consistessentially of, administering to a mammal having cancer, a recombinantAd comprising (a) an Ad genome from a first Ad strain and (b) at leastone hexon polypeptide from a one or more Ad strains, where one or moreof the hypervariable regions (HVRs) of the hexon polypeptide aredifferent from the HVRs encoded by the Ad genome and/or an Ad comprising(a) nucleic acid encoding a first hexon polypeptide and (b) a secondhexon polypeptide, where the amino acid sequence of the first hexonpolypeptide is different from the amino acid sequence of the secondhexon polypeptide. The mammal can be a human. The cancer can be prostatecancer, ovarian cancer, lung cancer, hepatocellular carcinoma,pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer,lymphoma, myeloma, lymphocytic leukemia, or myelogenous leukemia. Theadministering can include systemic or local administration (e.g.intravenous, intratumoral, intramuscular, intraorgan, intralymph nodeadministration).

It is demonstrated herein that Ad657 and variants thereof are able todeliver therapeutic genes to cells for expression of therapeuticpolypeptides. Thus, recombinant Ads, including chimeric Ads, can be usedas a local or systemic delivery vehicle for nucleic acid, vaccines,and/or oncolytic virotherapy for cancers.

An aspect of the invention relates to recombinant adenovirus (Ad)comprising (a) an Ad genome encoding hexon polypeptides from a first Adstrain and (b) a nucleic acid encoding at least one hexon polypeptidefrom one or more different Ad strains, wherein at least onehypervariable region (HVR) of the hexon polypeptide is different fromthe HVRs encoded by the Ad genome of the first Ad strain.

A further aspect of the invention relates to such a recombinant Ad,wherein the first Ad strain and the one or more different Ad strains areserotypically distinct.

A further aspect of the invention relates to such a recombinant Ad,wherein the first Ad strain is a human Ad6 strain, and wherein a secondAd strain is a human Ad57 strain.

A further aspect of the invention relates to such a recombinant Adfurther comprising a nucleic acid encoding a targeting polypeptide,antigen, enzyme, receptor, ligand or tag.

A further aspect of the invention relates to such a recombinant Ad,wherein the targeting polypeptide comprises an amino acid sequenceselected from SEQ ID NO: 1-41 and SEQ ID NO:46-47.

A further aspect of the invention relates to such a recombinant Ad,wherein said recombinant Ad is a replication competent Ad.

A further aspect of the invention relates to such a recombinant Ad,wherein the replication competent Ad is a single-cycle Ad orconditionally-replicating Ad (CRAd).

A further aspect of the invention relates to such a recombinantadenovirus (Ad) comprising (a) Ad capsid polypeptides from a first Adstrain and (b) at least one hexon polypeptide from one or more differentAd strains, wherein hypervariable regions (HVRs) of the hexonpolypeptide or capsid polypeptides are different from the HVRs or capsidpolypeptides of the first Ad strain.

A further aspect of the invention relates to such a recombinantadenovirus (Ad) comprising (a) a nucleic acid encoding a first hexonpolypeptide and (b) a nucleic acid encoding a second hexon polypeptide,wherein the amino acid sequence of the first hexon polypeptide isdifferent from the amino acid sequence of the second hexon polypeptide.

A further aspect of the invention relates to such a recombinant Ad,wherein the nucleic acid encoding at least one hexon polypeptide isdifferent from the amino acid sequence of a hypervariable region of saidsecond hexon polypeptide.

A further aspect of the invention relates to such a recombinant Ad,wherein said nucleic acid is from a first Ad strain, and wherein saidsecond hexon polypeptide is from a second Ad strain.

A further aspect of the invention relates to such a recombinant Ad,wherein said first Ad strain is a first human Ad strain, and whereinsaid second Ad strain is a second human Ad strain different from saidfirst human Ad strain.

A further aspect of the invention relates to such a recombinant Ad,wherein said first Ad strain and said second Ad strain are serotypicallydistinct.

A further aspect of the invention relates to such a recombinant Ad,wherein said first Ad strain is a human Ad6 strain, and wherein saidsecond Ad strain is a human Ad57 strain.

A further aspect of the invention relates to such a recombinant Ad,further comprising a targeting polypeptide.

A further aspect of the invention relates to such a recombinant Ad,wherein said targeting polypeptide comprises an amino acid sequenceTARGEHKEEELI (SEQ ID NO:1).

A further aspect of the invention relates to such a recombinant Ad,wherein said recombinant Ad is a replication competent Ad.

A further aspect of the invention relates to such a recombinant Ad,wherein said replication competent Ad is a single-cycle Ad orconditionally-replicating Ad (CRAd).

A further aspect of the invention relates to a method for treating amammal having cancer, wherein said method comprises administering, tosaid mammal, a recombinant adenovirus (Ad) as described herein.

A further aspect of the invention relates to such a method, wherein saidcancer is selected from the group consisting of prostate cancer, ovariancancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidneycancer, melanoma, brain cancer, colon cancer, lymphoma, myeloma,lymphocytic leukemia, and myelogenous leukemia.

A further aspect of the invention relates to such a method, wherein saidadministering comprises systemic administration.

A further aspect of the invention relates to such a method, wherein insystemic administration comprises intramuscular, intranasal, orintravenous administration.

A further aspect of the invention relates to such a method, wherein saidadministering comprises local administration.

A further aspect of the invention relates to such a method, wherein saidlocal administration comprises intratumoral injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a translation of context-specific peptides from phage toadenovirus. A) Diagram of a phage display library containing the Ad5fiber HI β sheets that structurally constrain a random 12-mer peptidelibrary. Shown below is a depiction of the structurally similar sitebetween the β7 and β8 sheets in the Ad5 HVR5 hexon. B) Primary aminoacid alignments of 12.51 and 12.52 in the HI library and their locationwhen inserted into HVR5 of hexon. C) Representation of Ad5 GFP-Lucexpressing viruses modified with the peptides.

FIG. 2 shows in vitro transduction A) GFP expression by fluorescentmicroscopy of C2C12 cells infected with 10⁴ vp/cell of the indicatedvectors 2 days after infection. B) Luciferase activity from C2C12 cells2 days after infection with varied MOIs of the indicated Ads.

FIG. 3 shows in vivo transduction in mice. A) Luciferase imaging of mice1 day after injection by the intravenous (IV) or intramuscular (IM)routes. IM injected mice received 10⁹ vp into each quadricep. IVinjected mice received 10¹⁰ vp by tail vein. B) Quantitation ofluciferase activity by imaging on the indicated days after injection. *p<0.05 by one way ANOVA. *** p<0.001 by one way ANOVA.

FIG. 4 shows in vivo transduction in hamsters. A) Luciferase activity inthe muscles of hamsters 1 day after injection with 10¹⁰ vp by the IMroute. ** p<0.01 by T test.

FIG. 5 shows gene-based immune responses 16 weeks after single IMimmunization. Mice from FIG. 3 were bled 16 weeks after IM injection andtheir sera were analyzed in serial dilutions by ELISA to detectantibodies against GFP protein. * p<0.05, ** p<0.01, *** p<0.001 byone-way ANOVA. **** p<0.0001 by one-way ANOVA. All Ad-injected micegenerated significant anti-GFP antibodies when compared to the PBS groupat sera dilutions of 1:10,000 to 1:1000. Comparisons of Ad5-GL-HVR-12.51and 12.52 to Ad5-GL are shown with black asterisks. Comparison betweenAd5-GL-HVR-12.51 and Ad5-GL-HVR-12.52 are shown with a gray asterisk. Agray dashed and dotted line at OD 0.06 shows the 95% confidence intervaldiscriminating antibodies that are different from the PBS group.

FIG. 6 shows a phylogenetic tree of whole genome sequences of humanadenovirus serotypes.

FIG. 7 shows an alignment of Ad5, 6, and 57 showing variation in hexonand E3 regions. (A) A Pustell DNA alignment of the genomes of Ad6 andAd57. Boxes indicate hexon and E3 regions where variation is highestbetween the two viruses. (B) ClustalW amino acid alignment of thehypervariable region in hexon proteins from Ad5, Ad6, and Ad57.Alignments were performed on MacVector.

FIG. 8 shows a cartoon of the construction of Ad657 by replacement ofthe Ad6 HVRs with Ad57 HVRs. Abbreviation: HVRs, hypervariable regions.

FIG. 9 shows in vitro oncolytic activity. LNCaP and DU145 cells weretreated with the indicated viruses with the indicated vp/cell for 5days. The cells were stained with crystal violet and cell viability wasmeasured by OD595. Cell viability (%) was calculated by dividing the ODof the samples by the mean OD of untreated control cells on the same96-well plate and multiplying this number by 100. (A) LNCaP cellkilling. (B) DU145 killing. Abbreviation: vp, viral particle.

FIG. 10 shows effects of oncolytic Ads on liver damage. C57BL/6 mice(n=6 per group) were injected with 1011 vp of each virus by tail vein.(A) Kaplan-Meier survival. (B) Blood was drawn for ALT measurements 3days after injection (****p<0.001 by ANOVA). Abbreviations: ALT, alanineaminotransferase; vp, viral particle.

FIG. 11 shows anticancer activity of Ad6 and Ad657 in DU145 tumorxenografts in nude mice after single i.v. administration. Nude mice (n=9per group) bearing established DU145 tumors were injected i.v. with asingle dose of 3×10¹⁰ vp of the indicated viruses or with PBS. (A)Effect of a single i.v. injection on tumor growth. Tumor dimensions weremeasured with calipers and tumor volume was calculated aswidth²×length×½. The data are shown as mean±SE. *p<0.05, ****p=0.0001 byANOVA or by T-test as described in the text. Black asterisks with ablack arrow pointing up indicate the statistical difference between theAd6 group and the PBS group on a selected day described in the text.Gray asterisks and an arrow pointing up indicate differences between theAd657 group and the PBS group on the indicated day. The shadowed whiteasterisks with a gray arrow pointing down indicates the statisticaldifference between the Ad6 and Ad657 groups on the indicated day. (B)Effect of a single i.v. injection on survival. Animals were euthanizedwhen the tumor volume reached 2000 μL or when other sacrifice criteriawere met (e.g., ulceration) and Kaplan-Meier survival curves wereplotted (*p<0.05, **p<0.01 by log-rank analysis). Abbreviation: i.v.,intravenous.

FIG. 12 shows luciferase imaging nude mice. Four days after single i.v.injection of 3×10¹⁰ vp of Ad6 and Ad657-GFP-Luc with deletions of partof 12.5K, 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ), and 14.7Kand a partial deletion of E4 34K. Abbreviations: i.v., intravenous; vp,viral particle.

FIG. 13 shows luciferase imaging nude mice. Fourteen days after singlei.v. injection of 3×10¹⁰ vp of Ad657-GFP-Luc. Abbreviations: i.v.,intravenous; vp, viral particle.

FIG. 14 shows plasma HIV Env binding titers. Immunizations withdifferent SC-Ads and gp140 proteins are shown above the graph with largearrows. Midpoint F8 gp140 binding titers by ELISA are shown for eachanimal before and after each immunization. The dashed line indicates theminimal detection limit for antibodies in this assay. Symbols arescattered in the x direction at each time point to allow individualmeasurements to be observed. SC-Ad6-Ebov is a negative control Advaccine. This group of animals was not boosted with gp140. * p<0.05, **p<0.01, *** p<0.001, **** p<0.0001 by one way ANOVA in comparison to theSC-Ad6-Ebov group.

FIG. 15 shows plasma HIV neutralization titers. Neutralization of theindicated viruses was performed using the TZM-bl neutralization assay.All values were calculated as compared to virus-only wells. Each dotrepresents the mean value for each animal.

FIG. 16 shows plasma ADCC activity. Plasma samples were tested withCD16-KHYG-1 effector cells to kill CEM.NKR.CCR5.CD4+-Luc, target cellsinfected with SHIVSF162P3. Each dot represents the mean value for eachanimal. * p<0.05, *** p<0.001, **** p<0.0001 by one-way ANOVA vs. theSC-Ad6-Ebov group.

FIG. 17 shows mucosal ADCC activity. Vaginal wash and saliva sampleswere tested with CD16-KHYG-1 effector cells to killCEM.NKR.CCR5.CD4+-Luc, target cells infected with SHIVSF162P3. Each dotrepresents the mean value for each animal. * p<0.05 by one-way ANOVA vs.the SC-Ad6-Ebov group.

FIG. 18 shows IFN-γ Secreting Cells from PBMCs and Lymph Nodes. PBMCsand lymph node cells were analyzed by ELISPOT by staining for IFN-γ.Anti-Env indicates cells that were stimulated with conserved HIV Envpeptides, and SC-Ads. The total number of spot forming cells (SFCs) ineach of the stimulated wells were counted and adjusted to control mediumas background. Each dot represents the mean value for each animal. *p<0.05 by one-way ANOVA.

FIG. 19 shows mucosal T cell trafficking and activation. T cells wereharvested from rectal biopsies collected after the second protein boostand analyzed by flow cytometry for CD4, CD8, α4β7 integrin, CD69, andFoxP3. Each dot represents the mean value for each animal.

FIG. 20 shows Tfh cell response in the blood and in lymph nodes. PBMCsand lymph node cells collected at week 40 were stimulated with HIV-1 Envprotein and then examined for co-expression of CD3+, CD4+, CXCR5+, andIL-21. Each dot represents the mean value for each animal. * p<0.05 byone-way ANOVA.

FIG. 21 shows protection against repeated rectal SHIV_(SF162P3)challenge. The indicated groups were challenged rectally with 4.3 TCID50(on rhesus PBMCs) of SHIVSF162P3 on a weekly basis. Plasma samples wereanalyzed for SHIV viral RNA copies. Animals with RNA copies above 10were considered infected and the number of challenges required to infectthat animal were used as events for Kaplan-Meier survival analysis.

FIG. 22 shows SHIV_(SF162P3) acquisition and viral loads. A) Animalsfrom FIG. 8 were grouped by their initial SC-Ad priming route (IM or IN)yielding groups of 8 and Kaplan-Meier analysis was performed. B) PlasmaSHIVSF162P3 viral RNA levels over the course of the challenge study.

FIG. 23 shows SHIV viral load in tissues. RNA from PBMCs and post-mortemtissues were collected and qPCR was performed to detect analyzed forSHIV viral RNA.

FIG. 24 shows single-cycle adenovirus vaccines used in Example 7. A)Cartoon of SC-Ad serotypes 6 and 657 carrying F8 and G4 clade B HIVenvelope genes. B) Alignment of clade C Ad hexons including the Ad6 and57 hexons displayed on vaccines.

FIG. 25 shows saliva and vaginal HIV env binding titrations. ELISA OD450levels are shown for the indicated samples at the indicated dilutionswhen tested against F8. The low level of antibodies in these mucosalsamples prevent reaching saturation of the assay. For this reason, EC50values cannot be reliably calculated for most animals. Rhesus macaqueRh13-091 in the IN-IM-IM group was the only animal in which an EC50could be calculated (EC50=4580). Similar results were observed in ELISAsusing SF162 gp140.

FIG. 26 shows Ad657 expressing antigen genes from hepatitis C andcytomegalovirus (CMV) gB generating in vivo cytotoxic T lymphocyte (CTL)activity. Shown is killing of hepatitis C peptide-loaded target cells inmice vaccinated with Ad657-HCV rather than CMV gB.

FIG. 27 shows expression of human GMCSF from Ad657 inducingproliferation of GMCSF-dependent TF-1 human erythroblasts.

FIG. 28 is a graph showing Ad6 single IV injection vs. A549 lung tumors.

FIG. 29 is a graph showing Ad6 single IV or IT injection vs. Panc1pancreatic tumors.

FIG. 30 is a graph showing Ad6 single IV injection vs. kidney cancer inimmune competent hamsters.

FIG. 31 is a graph showing luciferase activity in B16 melanoma and A549lung tumor/cancer cells by Ads displaying 12.51 cell binding peptides inHVRS of the hexon.

FIG. 32 is a graph showing luciferase activity in hepatocellularcarcinoma and kidney cancer by Ads displaying 12.51 cell bindingpeptides in HVRS of the hexon.

FIG. 33 shows a cartoon combining the insertion of individual HVRs fromdifferent Ad serotypes with the insertion of cell targeting/detargetingpeptides or novel amino acids such as cysteine into the hexon fortargeted chemical modification and shielding. Depicted are chimeric HVRconstructs that combine different HVRs from different Ad serotypes tomodulate natural interactions with cells and blood factors improvepharmacology combined with insertion of cell binding and celldetargeting peptides in different HVRs to change cell entry and cellavoidance. If one HVR is substituted from 100 Ads, this would create 100different hexon chimeras. If all 7 HVRs each receive a different Ad HVR,this combinatorial library would equal 7¹⁰⁰ variants. If one 1 peptidewere introduced into 7 HVRs this would equal 7×7¹⁰⁰ variants. If 10different peptides were introduced into 7 HVRs, this would equal10×7×7¹⁰⁰ variants, etc.

FIG. 34 shows plasmid maps for representative combinatorial hexons andpeptide combinations. Shown are hexons with HVR1 from Ad6 and HVRs 2-7from Ad57 as well as insertions of cell targeting peptides intoindividual HVRs.

FIG. 35 shows chimeric HVR constructs that combine different HVRs fromdifferent Ad serotypes to modulate natural interactions with cells andblood factors improve pharmacology combined with insertion of cellbinding and cell detargeting peptides in different HVRs to change cellentry and cell avoidance. In this example, a single cysteine amino acidis inserted into the HVR1 and HVR5 of Ad657 to modulate pharmacology andallow targeted conjugation of polymers like polyethylene glycol or othermoieties like imaging agents like fluorophores.

FIG. 36 shows conjugation of polyethylene glycol (PEG) to Ad657-HVR1-C.A) SDS-PAGE of Ad proteins with and without PEGylation. Arrows show sizeincreases due to chemical attachment of PEG to hexon. B) Effects oftargeted PEGylation by maleimide-PEG and non-targeting NHS-PEG on virusinfection.

FIG. 37 shows conjugation of polyethylene glycol (PEG) to Ad657-HVR5-C.A) SDS-PAGE of Ad proteins with and without PEGylation. B) Near infraredimaging of SDS-PAGE of Ad proteins with and without PEGylation and withand without the near infrared fluorescence imaging tag IR800. C) In vivotransduction after intraperitoneal injection of maleimide-PEGylatedAd657-HVR5-C by luciferase imaging.

FIG. 38 shows luciferase imaging of nude mice. A) 1, 4, 7, 14, 28, and42 days after single I.V. injection of Ad6 treatment vs. distant DU145prostate tumors. B) 3, 7, and 19 days after I.V. injection ofreplicating Ad5-GFPLUC into mice bearing LNCaP prostate tumors.

FIG. 39 shows a schematic of cancer-specific conditionally-replicatingAds (CRAds) dl1101+dl1107 having a modification in the E1A gene.

FIG. 40 is a graph showing that Ad6 and Ad657 can both be used as CRAdsfor targeted cancer therapy.

FIG. 41 is a schematic showing Ad therapeutic cycles. A) A schematic ofserotype-switching with Ads. B) A schematic of an exemplary therapeuticcycle where Ad6 and Ad657 can be used for multiple rounds of treatmentby serotype-switching in combination with covalent polymer conjugation.

FIG. 42 demonstrates serotype-switching and on-target luciferaseactivity in the DU145 prostate tumors after a single IV injection of Ad6and Ad6-F35 with deletions in E3A genes (12.5K, 6.7K, 19K, 11.6K), butretention of E3B genes (10.4K, 14.5K, and 14.7K) and retention of E434K. Mice whose tumors resisted prior single IV injection with Ad657 andCRAd657 both with intact E3 genes were injected with the indicatedvectors by single IV injection.

FIG. 43 is a schematic of a replication competent Ad (RC-Ad), wherein E1expression is controlled by the native E1 promoter; a variantCRAd-Probasin-E1A (Ad-PB), wherein E1 expression is controlled byprostate-specific probasin promoter; CRAd-dl1101, wherein p300 pathwaybinding ablated, susceptible to IFN pathway in normal cells;CRAd-dl1107, wherein pRB binding ablated allows virus to kill cancercells with RB pathway disruptions, but is repressed in RB+ normal cells;CRAd-dl1101/07, wherein p300 pathway binding ablated, susceptible to IFNpathway pRB binding ablated allows virus to kill cancer cells with RBpathway disruptions, but is repressed in RB+ normal cells.

FIG. 44 (A and B) shows the effect of infection withreplication-competent Ad5, Ad6, Ad657 on non-cancerous cells andmodification of Ad6 and Ad657 to be conditionally-replicating Ads(CRAds).

FIG. 45 demonstrates killing of cancerous cells by replication-competentAd5, Ad6, Ad657, and the indicated CRAds.

FIG. 46 demonstrates modification of Ad6 and Ad657 to beconditionally-replicating Ads (CRAds).

FIG. 47 demonstrates modification of Ad6 and Ad657 to beconditionally-replicating Ads (CRAds).

FIG. 48 demonstrates in vivo effects of replication-competent Ad6 or theindicated CRAds on growth of DU145 tumors in mice.

FIG. 49 demonstrates in vivo effects of replication-competent Ad657 andconditionally-replicating Ad657-dl1101/07 both with intact E3 regions invivo after a single intravenous injection in mice bearing human prostatetumors.

FIG. 50 demonstrates that PEGylation de-targets adenovirus to liver invivo.

FIG. 51 demonstrates modification of Ad657 with the shorter fiber fromchimpanzee AdC68 and the addition of a codon-wobbled E4 34.K genechanges in vitro efficacy.

FIG. 52 demonstrates 6/57/6 virus killing human prostate cancer cellswith and without CRAd modifications.

FIG. 53 demonstrates the production of antibody responses against thehuman cancer antigen folate receptor alpha after a single intramuscularimmunization of BALB/c mice by CRAd657-dl1101/07-FOLR with an intact E3region.

FIG. 54 depicts sites on Ad HVRs which may be modified, for example, byPEGylation or “BAPylation” with biotin acceptor peptides (BAPs).

FIG. 55 is a schematic of variants of Ads having mutations in the E1protein to convert the virus to a conditionally-replicating Ad (CRAd).

FIG. 56 shows amino acid sequences of the N-terminal portion of thewild-type E1A polypeptide and the E1A N-terminus of the CRAd variants,dl1101, dl1107 and dl1101/1107.

FIG. 57 shows as schematic of different E3 immune evasion genes in Adspecies C exemplar Ad6 and Ad species D exemplar Ad26. Both Ads expresssize and sequence variants of E3 12.5K, 6.7K, 19K, 10.4K (RIDα), 14.5K(RIDβ), and 14.7K genes, as well as a depiction of the functions ofthese E3 encoded proteins.

FIG. 58 demonstrates the effects of PEGylation and E3 deletion ononcolytic viral anti-tumor activity by Ad6-Luc viruses inimmunocompetent hamsters. Ad6-Luc and Ad6-Luc-20K PEG both have all E3genes and E4 34K intact. Ad6-deltaE3-Luc has partial deletion of E312.5K and E4 34K and full deletion of E3 6.7K, 19K, 11.6K (ADP), 10.4K(RIDα), 14.5K (RIDβ), and 14.7K genes. Oncolytic efficacy is lost inthis immunocompetent animal model when these immune evasion genes arenot present in oncolytic adenovirus.

FIG. 59 is a plasmid map of Ad657 with partial deletion of E3 12.5K andE4 34K and full deletion of E3 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα),14.5K (RIDβ), and 14.7K genes.

FIG. 60 depicts CRAd 657 constructs with and without dl1101/1107 CRAdmodifications and with and without deletions of selected E3 immuneevasion genes.

FIG. 61 depicts CRAd657 with E3 insertion site. These are with andwithout dl1101/1107 CRAd modifications described herein and with andwithout E3 immune evasion modifications.

FIG. 62 depicts CRAd657+/−Ad35 Fiber or Chimpanzee C68 Fiber+/−K7peptide. These are with and without dl1101/1107 CRAd modificationsdescribed in previous slides and with and without E3 immune evasionmodifications. In some cases, a codon-wobbled E4 34K gene is includedafter E4 and before fiber to compensate for E4 34K partial deletion whendeleted E3B genes.

FIG. 63 depicts CRAd657+/−Ad35 Fiber or Chimpanzee C68 Fiber+/−K7peptide. These are with and without dl1101/1107 CRAd modificationsdescribed in previous slides and with and without E3 immune evasionmodifications.

FIG. 64 depicts CRAd657+/−Ad35 Fiber or Chimpanzee C68 Fiber+/−K7peptide Expressing Folate Receptor alpha.

FIG. 65 depicts CRAd657+/−Ad35 Fiber or Chimpanzee C68 Fiber+/−K7peptide Expressing Granulocyte Macrophage Colony Stimulating Factor(GMCSF).

FIG. 66 depicts CRAd657+/−Ad35 Fiber or Chimpanzee C68 Fiber+/−K7peptide Expressing 4-1BBL or GMCSF or IL21 or CD40L and combinations inone virus.

FIG. 67 depicts Ad6/57 with Ad6 HVR1 and Ad57 HVRs2-7+/−Ad35 Fiber orChimpanzee C68 Fiber+/−K7 peptide.

FIG. 68 depicts Ad6/57/6 with Ad6 HVR1, Ad57 HVRs2-6, Ad6 HVR7+/−Ad35Fiber or Chimpanzee C68 Fiber+/−K7 peptide.

FIG. 69 depicts Ad6/57/6 with Ad6 HVR1, Ad57 HVRs2-6, Ad6 HVR7+/−Ad35Fiber or Chimpanzee C68 Fiber+/−K7 peptide expressing GFPLuciferase.

FIG. 70 shows luciferase imaging after serotype switching. Mice bearingLNCaP prostate tumors on their flanks were treated by a single IVinjection of Ad657 or CRAd657. Mice with residual tumors 5 months aftersingle IV injection were treated by serotype-switching of the indicatedAd6/57/6 variants expressing GFPLuciferase with and without variantfibers and a codon-optimized E4 34K gene. The indicated Ad6/57/6variants include Ad6/57/6 virus having different fiber modificationsincluding an added 7 lysine on fiber (K7), chimpanzee C68 fiber graftedonto Ad6 fiber after its KKTK flexibility domain and with an Ad35 fiber.Mice were imaged for luciferase activity 7 days later.

FIG. 71 shows A549 human lung cancer cells which were treated with theindicated viral particles (vp) per cell of Ad6/57/6 with and withoutvariant fibers and a codon-optimized E4 34K gene. 7 days later, thecells were stained with crystal violet and the wells were analyzed in aplate reader. High OD indicates the presence of viable cells. Low ODindicates death and loss of adherent cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods and materials for nucleic acid delivery,vaccination, and/or treating cancer. For example, this inventionprovides methods and materials for nucleic acid delivery ofproteins/polypeptides, vaccination, and/or treating cancer using one ormore recombinant Ads (e.g., Ad657 and variants thereof) as an oncolyticagent.

An adenovirus icosahedron is made up of 720 copies of its hexon protein.The virus does not use this protein to bind receptors, but thisnano-lattice of repeating proteins provides a matrix for interactions(e.g., natural interactions and unnatural interactions) with proteins,cells, and drugs. Antibodies that can neutralize Ads can targethypervariable regions (HVRs) of the hexon polypeptide on an Ads.

In some cases, this invention provides recombinant Ads having oncolyticanti-cancer activity. For example, a recombinant Ad can be derived froma first Ad and can include hexon HVRs from one or more different Ads.The HVRs may be derived from any species C Ads, for example Ad1, Ad2,Ad5, Ad6 and Ad57. In an embodiment, a recombinant Ad can be derivedfrom a first Ad and can include one or more hexon HVRs from at least oneother Ad, wherein at least one hexon HVR is different from the HVR(s) ofthe first Ad. The first Ad strain can be a human Ad6 strain, and thesecond Ad strain can be a human Ad57 strain. Hexon shuttle plasmid maps(FIG. 34 ) show the combination of the insertion of individual HVRs fromdifferent Ad serotypes with the insertion of cell targeting/detargetingpeptides or novel amino acids such as cysteine into the hexon fortargeted chemical modification and shielding. In an embodiment, therecombinant Ads comprise amino acid substitutions, for example,substitution of cysteines into polypeptides, and modifications such asPEGylation and BAPylation. The ability to target polymer and otherchemical modifications to cysteines inserted in Ad657 hexon isdemonstrated herein.

Ad657 as an oncolytic against human prostate cancer is demonstrated. TheAd6 HVRs were replaced with those from Ad57 to generate a chimericspecies C oncolytic virus called Ad657. Ad657 and Ad6 were tested assystemic oncolytic therapies by single i.v. injection in nude micebearing human cancerous tumors. Ad657 may be used as a local or systemiconcolytic virotherapy for cancers. These data also demonstratesurprising effects of serotype-switching with oncolytic species C Ads.

In some cases, this invention provides methods for using one or morerecombinant Ads provided herein to treat a mammal having, or at risk ofhaving, cancer, an infectious disease, and/or a genetic disease. Forexample, one or more recombinant Ads can be administered to a mammalhaving, or at risk of having, cancer to reduce the number of cancercells (e.g., by infecting and killing cancer cells) in the mammal (e.g.,a human). For example, one or more recombinant Ads can be administeredto a mammal having, or at risk of having, cancer to stimulateanti-cancer immune responses in the mammal (e.g., a human).

In some cases, recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657 andvariants thereof) are not destroyed by a mammal's immune system. Forexample, a recombinant Ad is not destroyed by antigen presenting cells(APCs), macrophages, and/or other immune cells in a mammal that therecombinant Ad is administered to.

In some cases, recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657 andvariants thereof) can be administered for multiple (e.g., two or more)rounds of treatment. For example, a first recombinant Ad describedherein can avoid antibodies that can neutralize a second recombinant Addescribed herein, and vice versa. In cases where a mammal having canceris treated with one or more recombinant Ads described herein, the mammalcan be administered a first round of treatment with a first recombinantAd and can subsequently be administered a second round of treatment witha second recombinant Ad.

In some cases, recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657 andvariants thereof) can be replication competent Ads (RC-Ads). Forexample, a RC-Ad can be a RC-Ad that includes a nucleic acid encoding anE1 polypeptide (e.g., an E1+RC-Ad). For example, a RC-Ad can be asingle-cycle Ad (SC-Ad) that includes a deletion of one or more nucleicacids encoding one or more polypeptides associated with the productionof infectious viral progeny (e.g., pIIIa and E3). For example, a RC-Adcan be a conditionally-replicating Ad (CRAd). Examples of single-cycleAds and how to make and use them are provided elsewhere (InternationalPatent Application Publication No. WO2009/111738).

In some cases, recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657 andvariants thereof) can be replication defective Ads (RD-Ads). Forexample, a RD-Ad can be a RD-Ad that includes a deletion of a nucleicacid encoding an E1 polypeptide (e.g., an E1-deleted RD-Ad).

It is demonstrated in the examples herein that CRAd 657 and variantsthereof are conditionally-replicating Ads (CRAds) in cancerous cells andthat infection of cells with CRAd 657 and variants thereof reduces cellviability and tumor volume. Thus, CRAd 657 and variants thereof may beused as a local or systemic oncolytic virotherapy in subjects withcancer.

What is more, it is demonstrated that CRAds can be used for expressionof antigens and used as a vaccine for vaccinating against viruses, forexample, against Human Immunodeficiency Virus (HIV), Human PapillomaVirus (HPV) and Hepatitis C Virus (HCV).

In some cases, recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657 andvariants thereof) can bind to a cell surface receptor (e.g., tofacilitate viral entry to a cell). For example, a recombinant Addescribed herein can bind to coxsackie-adenovirus receptors (CARs)and/or Fc receptors (e.g., FcμR and FcγR), complement receptors (e.g.,CR3 and/or C2qR).

In an aspect of the invention, CRAds may comprise nucleic acids encodingpolypeptides heterologous to the Ad, for example, antigens, cell surfacereceptors, cell targeting polypeptides and the like. For example,CRAd-657-dl1101/1107-FolR is a recombinant Ad comprising intact E3 andexpressing the human folate receptor alpha. It is demonstrated hereinthat CRAds may be used to generate antibodies against known cancerantigens, for example, folate receptor alpha.

In some cases, recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657s) canavoid binding (e.g., do not bind) to a scavenger receptor (e.g., tofacilitate viral entry to a cell). For example, a recombinant Addescribed herein avoid binding to a SREC receptors and/or SR-Areceptors.

In some cases, recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657s) canavoid phagocytosis.

In some cases, recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657s) arenon-pathogenic (e.g., to a mammal being treated as described herein).

In some cases, recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657s) caninfect dividing cells (e.g., can infect only dividing cells).

A recombinant Ad described herein can be any appropriate recombinant Ad(e.g., a recombinant Ad having oncolytic anti-cancer activity) generatedby recombinant DNA technology and methods known to those skilled in theart. A recombinant Ad can be any Ad generated by recombining material(e.g., nucleic acid and/or polypeptide) from any organism other than theAd from which the recombinant Ad is derived. For example, a recombinantAd can include one or more materials that do not naturally occur in thatAd (e.g., do no naturally occur in that Ad prior to recombination). Insome cases, a recombinant Ad provided herein can be a chimeric Ad (e.g.,can include viral elements from two or more (e.g., two, three, four,five, or more) different Ad genomes).

These embodiments have been applied also in the context of Ads whichcombine different HVRs from different Ads (i.e., shuffling HVRs). Forexample, HVR1 of Ad6 with HVRs 2-7 of Ad57 or HVR1 and 7 of Ad6 withHVRs 2-6 of Ad57, or HVRs 1 and 7 from Ad6 and HVRs 2-6 from Ad657.

Nucleic acid and/or polypeptides that do not naturally occur in the Adcan be from any appropriate source. In some cases, a nucleic acid and/ora polypeptide that does not naturally occur in that Ad can be from anon-viral organism. In some cases, a nucleic acid and/or a polypeptidethat does not naturally occur in that Ad can be from a virus other thanan Ad. In some cases, a nucleic acid and/or a polypeptide that does notnaturally occur in that Ad can be from an Ad obtained from a differentspecies. In some cases, a nucleic acid and/or a polypeptide that doesnot naturally occur in that Ad can be from a different strain of Ad(e.g., serotypically distinct strains). In some cases, a nucleic acidand/or a polypeptide that does not naturally occur in that Ad can be asynthetic nucleic acid and/or a synthetic polypeptide.

A recombinant Ad described herein (e.g., a recombinant Ad havingoncolytic anti-cancer activity such as a recombinant Ad657) can bederived from (e.g., can include a genomic backbone from) any appropriateAd. In some cases, a recombinant Ad described herein can be derived froman Ad having low seroprevalence. For example, 50% or fewer (e.g., 50%,45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or fewer) of mammals (e.g.,human) can have been exposed to an Ad from which a recombinant Addescribed herein is derived. With regard to seroprevalence, species Cadenoviruses, Ad6 and Ad657 have lower prevalence than archetype Ad5virus. In some cases, a recombinant Ad described herein can be derivedfrom an Ad having reduced or eliminated side effects (e.g., phagocytosisand liver damage). A recombinant Ad can be derived from an Ad isolatedfrom any appropriate species of animal. For example, Ads can be isolatedfrom humans, non-human primates (e.g., monkeys such as Old World monkeyspecies like rhesus macaques), fish, frogs, and snakes. In some cases, arecombinant Ad described herein can be derived from a human Ad (HAd orHAdV). A recombinant Ad can be derived from any species of Ad (e.g., A,B, C, D, E, F, or G). In some cases, a recombinant Ad described hereincan be derived from an Ad C species (e.g., a human Ad C species(HAd-C)). A recombinant Ad can be derived from any appropriate Adserotype (e.g., 2, 5, 6, or 57). In some cases, a recombinant Addescribed herein can be derived from an Ad serotype 6 (Ad6; e.g., ahuman Ad6).

In some cases, a recombinant Ad described herein (e.g., a recombinant Adhaving oncolytic anti-cancer activity such as a recombinant Ad657 andvariants thereof) can include an Ad genome containing one or moremodifications to one or more nucleic acids encoding a polypeptide (orfragments thereof) and/or one or more viral elements of the Ad genome.The one or more modifications can be any appropriate modification. Insome cases, a modification can be effective to inhibit the ability ofthe modified polypeptide to bind another polypeptide such as p300 and/orpRB. In some cases, a modification can be effective to neutralize one ormore interferon pathways. Examples of modifications that can be made toa nucleic acid encoding a polypeptide or to a viral element include,without limitation, substitutions, deletions, insertions, and mutations.

Ads, for example Ad657 and variants thereof, may be modified and retainall E1A genes, or modified to delete selected regions and functions oftheir encoded proteins.

FIG. 57 shows as schematic of different E3 immune evasion genes in Adspecies C exemplar Ad6 and Ad species D exemplar Ad26, as well as adepiction of the functions of these E3 encoded proteins. Both Adsexpress size and sequence variants of E3 12.5K, 6.7K, 19K, 10.4K (RIDα),14.5K (RIDβ), and 14.7K genes. 19K reduces display of MHC I and MICproteins on the cell surface to protect infected cells from T cells andNK cells. RID proteins protect infected cells from death-inducingligands (FAS, TRAIL, TNFR, and EGFR). 14.7K inhibits intrinsicactivation of apoptosis in infected cells. Species C Ads also expressthe 11.6K known as the adenovirus death protein (ADP). Over-expressionof ADP accelerates cell death, but overall cell death is equal. SpeciesD viruses also express two novel variants called 49K and 31K. Thesecreted form of 49K binds to CD46 on T cells and NK cells leading todown-regulation of these cells and less-efficient cell killing of cellsdeficient in class I MHC by NK cells. Ad657 plasmids have been modifiedto retain all native E3 immune evasion genes (12.5K, 6.7K, 19K, 11.6K(ADP), 10.4K (RIDα), 14.5K (RIDβ), and 14.7K) and E4 34K or to deleteselected regions. Ad657 and its variants are also modified with theaddition of 49K and 31K to provide these extra functions to thesespecies C viral platforms.

Ads, for example Ad657 and variants thereof, may be modified to retainall E3 immune evasion genes, or to delete selected regions and functionsof their encoded proteins. With respect to E3 mutations: 19kdownregulates MHCI and MIC proteins on infected cells; ADP overexpression accelerates cell death, but does not increase the number ofcells that are killed; 10k and 14k proteins (RIDα and RIDβ) combine toblock cell killing by extrinsic apoptosis proteins like FAS, TRAIL, TNF,TNFR, and EGFR; 14.7k protein inhibits intrinsic apoptosis signaling.

Retaining these E3 proteins may allow oncolytic to persist longer, anddeleting them may increase immune stimulation.

Data testing oncolytic efficacy suggests intact E3 mediates betterefficacy.

DE3 constructs have deleted part of 12.5k through and including 14.7k,DE3A constructs have deleted part of E3 12.5k through and including 19k,and DE3ADP constructs have deleted part of E3 12.5k through andincluding ADP.

Surprisingly, deleting all E3 genes makes the oncolytic virus lesseffective in repressing tumor growth.

In an embodiment, the invention encompasses single-cycle adenovirus, forexample SC-Ad657 and variants thereof. Recombinant SC-Ad viruses withheterologous nucleic acids encoding polypeptides were evaluated for useas a vaccine. SC-Ad657 vaccines by themselves generated significantcirculating antibody titers against an HIV envelope protein after only asingle immunization.

Similarly, Ad657 carrying genes for hepatitis B and C antigens is ableto generate cytotoxic T lymphocyte (CTL) responses against hepatitis andcytomegalovirus.

Ad657 was modified by insertion of synthetic peptides from humanpapilloma virus into HVR5. In an embodiment, the amino acid sequence ofthe variant Ad657-HVR5-HPV hexon is defined in SEQ ID NO:57. Themodification allows display of this antigen for vaccine purposes as wellas retargeting by binding to proteins that interact with HPV peptides.

In a further embodiment, expression of Human Granulocyte-MacrophageColony Stimulating Factor (GMCSF) by Ad657 is demonstrated herein.

Thus, from the examples herein, it is demonstrated that recombinant Ads,for example Ad657 and variants thereof, may be utilized for expressionof heterologous proteins, for example, polypeptide antigens and celltargeting polypeptides.

In some cases, a recombinant Ad described herein (e.g., a recombinant Adhaving oncolytic anti-cancer activity such as a recombinant Ad657) caninclude an Ad genome containing one or more substitutions. For example,one or more nucleic acids encoding a polypeptide (or fragments thereof)and/or one or more viral elements encoded by the Ad genome can besubstituted. A substitution can be any appropriate substitution. In somecases, one or more nucleic acids encoding a capsid polypeptide of agenome of a first Ad can be substituted with one or more nucleic acidsencoding a capsid polypeptide of a second Ad to generate a chimeric Ad.For example, when a recombinant Ad includes a genome from a first Adwhere a nucleic acid encoding a capsid polypeptide in the genome issubstituted for a nucleic acid encoding a capsid polypeptide from asecond Ad (e.g., an Ad different from the Ad backbone), the nucleic acidencoding a capsid polypeptide form the second Ad can express one or morecapsid polypeptides, and the expressed capsid polypeptide(s) can beincorporated into the capsid of the recombinant Ad. Examples of capsidpolypeptides include, without limitation, hexon polypeptides, fiberpolypeptides, penton base polypeptides, IIIc polypeptides, IXpolypeptides, and pVI polypeptides.

The Ad fiber protein is a complex of three apparently identical subunitswhich mediates the initial cell attachment step. The native Ad6 fiberprotein comprises the amino acid sequence set forth in SEQ ID NO:60 andbinds CAR.

In an aspect of the invention, fiber-modified recombinant and chimericAds having fiber proteins which are not native to the parental or“backbone” Ad were generated.

A chimeric Ad, AdF35 fiber chimera, has the amino acid sequence of SEQID NO:61 and is shorter than Ad5 and Ad6 fiber proteins and retargetsvirus to CD46.

A fiber-modified recombinant Ad, comprising K7 Fiber having the sequenceof SEQ ID NO:62, targets virus to heparin sulfate proteoglycans andnegative charges on cells.

A recombinant, chimeric Ad, 6/FC68 Fiber comprising the sequence of SEQID NO:63, is a chimeric Ad having a fiber protein from chimpanzeeadenovirus C68. The fiber protein is shorter than Ad5 or Ad6 fiberproteins and binds CAR.

A recombinant, chimeric Ad, 6/FC68-K7 Fiber comprising the sequence ofSEQ ID NO:64, is a chimeric Ad having a fiber protein from chimpanzeeadenovirus C68. The fiber protein is shorter than Ad5 or Ad6 fiberproteins. The 6/FC68-K7 Fiber binds CAR and is retargeted to heparinsulfate and negative charges.

A recombinant, chimeric Ad, 6/FC68-HI-K7 Fiber comprising the sequenceof SEQ ID NO:65, is a chimeric Ad having a fiber protein from chimpanzeeadenovirus C68. The fiber protein is shorter than Ad5 or Ad6 fiberproteins. The 6/FC68-HI-K7 Fiber binds CAR and is retargeted to heparinsulfate and negative charges.

In some cases, a recombinant Ad can include a genome from a first Adwhere a nucleic acid encoding a hexon polypeptide (e.g., HVRs of anucleic acid encoding a hexon polypeptide) in the genome is substitutedfor a nucleic acid encoding a hexon polypeptide (e.g., HVRs of a nucleicacid encoding a hexon polypeptide) from a second Ad. In some cases, arecombinant Ad described herein can include a genome from a first Adthat has one or more HVRs substituted for one or more HVRs from a secondAd. For example, a recombinant Ad can be a chimera, in particular Ad657(e.g., can include an Ad6 genome where the hexon HVRs are substitutedfor Ad57 hexon HVRs). In cases where a recombinant Ad includes a genomefrom a first Ad where a nucleic acid encoding a hexon polypeptide in thegenome is substituted for a nucleic acid encoding a hexon polypeptidefrom a second Ad, the recombinant Ad can include from about 1 to about720 hexon polypeptides from the second Ad. For example, when arecombinant Ad is an Ad657, the Ad657 can include an Ad6 genome and 720hexon polypeptides including Ad57 hexon HVRs.

In some cases, a recombinant Ad described herein (e.g., a recombinant Adhaving oncolytic anti-cancer activity such as a recombinant Ad657) caninclude an Ad genome containing one or more nucleic acid deletions. Anucleic acid deletion can be any appropriate nucleic acid deletion. Anucleic acid deletion can be a full deletion (e.g., deletion of anucleic acid encoding a polypeptide) or a partial deletion (e.g.,deletion of one or more nucleotides within a nucleic acid encoding apolypeptide). A nucleic acid deletion can reduce or eliminatetranscription and translation of a polypeptide encoded by the deletednucleic acid. Any appropriate nucleic acid can be deleted. In somecases, a nucleic acid encoding a polypeptide associated with productionof infectious progeny can be deleted. Examples of nucleic acids that canbe deleted and/or modified in a recombinant Ad described herein mayencode E1 (e.g., E1A and E1B), E2, E3, E4, pIIIA, fiber, E1B, andinclude viral enhancers and promoters. For example, a recombinant Addescribed herein (e.g., a recombinant Ad having oncolytic anti-canceractivity such as a recombinant Ad657) can include an Ad genomecontaining a deletion of one or more nucleotides within a nucleic acidencoding an E1 polypeptide. In some cases, a recombinant Ad describedherein can include one or more substitutions in a nucleic acid encodingan E1 polypeptide.

In particular embodiments, a recombinant Ad described herein is modifiedto comprise a probasin promoter comprising, for example, a nucleic acidof SEQ ID NO:48; a recombinant Ad described herein is modified tocomprise a dl1101 deletion in a nucleic acid encoding an E1 polypeptide;a recombinant Ad described herein is modified to comprise a dl1107deletion in a nucleic acid encoding an E1 polypeptide; a recombinant Addescribed herein is modified to comprise a dl1101 deletion and a dl1107deletion. See the examples herein and FIG. 56 for N-terminal amino acidsequences of the E1A polypeptide, for example, wild-type Ad E1A, andCRAd-657-dl1101, CRAd-657-dl1107 and CRAd-657-dl1101/1107 variants.

In an embodiment, a variant CRAd-657-dl1101/1107-FolR comprises intactE3 and expresses the human folate receptor alpha found on cancer cells.

In general, Ads may be modified to include CRAd modifications describedherein.

In some cases, a recombinant Ad described herein (e.g., a recombinant Adhaving oncolytic anti-cancer activity such as a recombinant Ad657) caninclude an Ad genome containing one or more nucleic acid insertions. Forexample, a nucleic acid insertion can include a nucleic acid encoding apolypeptide. A nucleic acid can be inserted into any appropriatelocation within a genome of a recombinant Ad described herein. In somecases, a nucleic acid encoding a polypeptide can be inserted into a HVR(e.g., HVR 5 loop) of a genome of a recombinant Ad described herein. Forexample, when a nucleic acid encoding a polypeptide is inserted into aHVR of a genome of a recombinant Ad described herein, the nucleic acidencoding a polypeptide can express one or more polypeptides, and theexpressed polypeptide(s) can be incorporated into the capsid of therecombinant Ad. In cases where a nucleic acid encoding a polypeptide isinserted into a HVR of a genome of a recombinant Ad described herein,the recombinant Ad can present from about 1 to about 720 polypeptidesencoded by the inserted nucleic acid on its surface. A nucleic acidinsertion can be nucleic acid encoding any appropriate polypeptide. Insome cases, a nucleic acid insertion can encode a polypeptide antigen.

In some cases, a nucleic acid insertion can encode a targetingpolypeptide. Examples of targeting polypeptides that can be included ina recombinant Ad described herein include, without limitation peptide12.51 (TARGEHKEEELI; SEQ ID NO:1), peptide 12.52 (LRQTGAASAVWG; SEQ IDNO:2), 12.53 (ARRADTQWRGLE; SEQ ID NO:3), VSV (GTWLNPGFPPQSCGYATVT; SEQID NO:4), RGD (CDCRGDCFC; SEQ ID NO:5), alpha4 integrin binding peptide(NMSLDVNRKA; SEQ ID NO:6), Met 3-4 (ISLSSHRATWVV; SEQ ID NO:7), L10.1F(WTMGLDQLRDSSWAHGGFSA; SEQ ID NO:8), L10.1RGDF (WTMGLDQLRGDSSWAHGGFS;SEQ ID NO:9), L10.2F (RSVSGTEWVPMNEQHRGAIW; SEQ ID NO:10), L10.5F(TELRTHTSKELTIRTAASSD; SEQ ID NO:11), S5.1 (DRAIGWQDKLYKLPLGSIHN; SEQ IDNO:12), DU9C.1 (MGSWEKAALWNRVSASSGGA; SEQ ID NO:13), DU9C.2(MAMGGKPERPADSDNVQVRG; SEQ ID NO:14), DU9A.7 (MASRGDAGEGSTQSNTNVPS; SEQID NO:15), XS.1 (GPEDTSRAPENQQKTFHRRW; SEQ ID NO:16), REDVmyc(MGREDVGEQKLISEEDLGGS; SEQ ID NO:17), RGD-4C (ACDCRGDCFCG; SEQ IDNO:18), REDV-4C (ACDCREDVCFCG; SEQ ID NO:19), SKBR5C1(GQIPITEPELCCVPWTEAFY; SEQ ID NO:20), 231R10.1 (PQPPNSTAHPNPHKAPPNTT;SEQ ID NO:21), HepaCD8 (VRWFPGGEWGVTHPESLPPP; SEQ ID NO:22), K20(KKKKKKKKKKKKKKKKKKK; SEQ ID NO:23), BAP (GLNDIFEAQKIEWH; SEQ ID NO:24),CALM BP (CAAARWKKAFIAVSAANRFKKIS; SEQ ID NO:25), EBV (EDPGFFNVEIPEFP;SEQ ID NO:26), #1-5 (GGHGRVLWPDGWFSLVGISP; SEQ ID NO:27), ##4*-5 (MARTVTANVPGMGEGMVVVPC; SEQ ID NO:28), 1-1 (GVSKRGLQCHDFISCSGVPW; SEQ IDNO:29), 1-2 (NQSIPKVAGDSKVFCWWCAL; SEQ ID NO:30), 1-3(QSTPPTKHLTIPRHLRNTLI; SEQ ID NO:31), 1-4 (DMSFQLVTPFLKALPTGWRG; SEQ IDNO:32), 1-5 (GGHGRVLWPDGWFSLVGISP; SEQ ID NO:33), 1-5con (FSLVGISP; SEQID NO:34), 1-6 (QIMMGPSLGYYMPSESIFAY; SEQ ID NO:35), 2-11(ISWDIWRWWYTSEDRDAGSA; SEQ ID NO:36), 2-14 (VWGMTTSDHQRKTERLDSPE; SEQ IDNO:37), 2-20 (MTSAQTSEKLKAETDRHTAE; SEQ ID NO:38), 2-9(MGSRSAVGDFESAEGSRRP; SEQ ID NO:39), 3b-6 (MGRTVQSGDGTPAQTQPSVN; SEQ IDNO:40), 4*-5 (MART VTANVPGMGEGMVVVP; SEQ ID NO:41), CLL peptides, PD-1,GLA polypeptides (e.g., Factor X), antigen genes, fusion proteins,fusogenic glycoproteins, single-chain antibodies, and capsid proteinsfrom other viruses. A targeting polypeptide can target any appropriatetype of cell. Examples of types of cells that can be targeted by atargeting polypeptide included in a recombinant Ad described hereininclude, without limitation, muscle cells (e.g., skeletal muscle cells),tumors, cancer cells, kidney cells, liver cells, mucosal cells,carbohydrates, and cell membranes.

This example demonstrates that peptides selected in a compatiblestructural context on phage libraries can be translated into the Adhexon protein. For example, for the 12.51 peptide, this insertion siteincreases muscle transduction while decreasing off target infection inthe liver. Thus, such a recombinant Ad which targets muscle tissue maybe used as a vector for gene-based muscle vaccination or for genetherapy application/delivery to the muscle.

In some cases, a nucleic acid insertion can detarget the virus (e.g., bydisrupting cell and protein interactions that occur on a given HVR). Insome cases, a nucleic acid insertion can encode a detectable label.Examples of detectable labels include, without limitation, fluorophores(e.g., green fluorescent protein (GFP), mCherry, and mBFP), and enzymes(e.g., luciferase, DNAses, proteases, transporters, and polymerases).

Also provided herein are expression vectors containing a recombinant Addescribed herein (e.g., a recombinant Ad having oncolytic anti-canceractivity such as a recombinant Ad657 and variants thereof). Expressionvectors can carry a recombinant Ad described herein into another cell(e.g., a cancer cell), where it can be replicated and/or expressed. Anexpression vector, also commonly referred to as an expression construct,is typically a plasmid or vector having an enhancer/promoter regioncontrolling expression of a specific nucleic acid. When introduced intoa cell, the expression vector can use cellular protein synthesismachinery to produce the virus in the cell. In some cases, expressionvectors containing recombinant Ads described herein can be viralvectors. For example, an expression vector containing a recombinant Addescribed herein can be a retroviral vector. In some cases, expressionvectors including a recombinant Ad described herein also can be designedto allow insertion of one or more transgenes (e.g., at a multi-cloningsite). For example, expression vectors including a recombinant Addescribed herein also can include a nucleic acid encoding a detectablelabel. Examples of detectable labels include, without limitation,fluorophores (e.g., green fluorescent protein (GFP), mCherry, and mBFP),and enzymes (e.g., luciferase, recombinases, nucleases, andtranscription factors).

This invention also provides methods and materials for using one or morerecombinant Ads described herein (e.g., recombinant Ads having oncolyticanti-cancer activity such as recombinant Ad657s). In some cases, arecombinant Ad provided herein can used for treating a mammal having, orat risk of having, cancer. For example, methods for treating a mammalhaving, or at risk of having, cancer can include administering one ormore recombinant Ads described herein to the mammal. In some cases,methods for treating a mammal having, or at risk of having, cancer caninclude administering one or more expression vectors that encode arecombinant Ad described herein or nucleic acid encoding a recombinantAd described herein to the mammal. In some cases, one or morerecombinant Ads described herein can be administered to a mammal toreduce the number of cancer cells in the mammal (e.g., suppress and/ordelay tumor growth) and/or to increase survival of the mammal.

Targeting a cancerous tumor by serotype-switching oncolytic adenovirusesis demonstrated. Mice bearing DU145 or LNCaP prostate tumors on theirflanks were treated one by intravenous (IV) injection with Ad657. Thesemice were treated a second time with alternate Ad6 or Ad6/57/6 oncolyticvirus variants with fiber modifications and expressing GFPLuciferase andluciferase activity was measured by imaging. Ad6 has Ad6 hexon and fiberthat targets CAR. Ad6-F35 has Ad6 hexon and the Ad35 fiber that targetsCD46. Ad6/57/6 has HVR1 and 7 from Ad6 and HVRs 2-6 from Ad57. Ad6/57/6viruses have Ad6 fiber, AdC68 fiber, or Ad35 fiber. These data in FIG. 9show the surprising ability to serotype-switch oncolytics with virusestargeting the tumor with lower off-target infection of the liver.

In another example of serotype-switching, mice bearing LNCaP prostatetumors on their flanks were treated by a single intravenous (IV)injection with Ad657 or CRAd657. These mice were treated a second time 5months later with alternate Ad6/57/6 oncolytic virus expressingGFPLuciferase and fiber variants K7 (with 7 lysines added), F35 (withthe Ad35 fiber), or KKTK-C68 (chimpanzee C68 fiber fused after the Ad6KKTK flexibility domain. KKTK-C68 virus also has an addedcodon-optimized E4 34K gene to enhance viral productivity. Luciferaseactivity was measured by imaging. All Ad6/57/6's have a hexon with HVR1and 7 from Ad6 and HVRs 2-6 from Ad57. Ad6/57/6 and KKTK-C68 have fibersthat targets CAR. Ad6/57/6-F35 has the Ad35 fiber that targets CD46. K7increases binding to negative charges on cells including binding heparinsulfate proteoglycans. FIG. 70 demonstrates the capability toserotype-switch oncolytics with viruses targeting a tumor with loweroff-target infection of the liver.

Any appropriate mammal having, or at risk of having, cancer, aninfectious disease, and/or a genetic disease can be treated as describedherein. For example, humans, non-human primates (e.g., monkeys), horses,bovine species, porcine species, dogs, cats, mice, rats, and feedanimals having cancer, an infectious disease, and/or a genetic diseasecan be treated for cancer as described herein. In some cases, a humanhaving cancer can be treated. In some cases, a mammal (e.g., a human)treated as described herein is not a natural host of an Ad used togenerate a recombinant Ad described herein (e.g., a recombinant Adhaving oncolytic anti-cancer activity such as a recombinant Ad657). Forexample, a human being treated with a recombinant Ad657 described hereincan lack any pre-existing adaptive immunity to Ad6.

A mammal having any type of cancer can be treated as described herein.In some cases, a cancer can include one or more solid tumors. In somecases, a cancer can be a blood cancer. Examples of cancers that can betreated as described herein include, without limitation, prostatecancer, ovarian cancer, lung cancer, hepatocellular carcinoma,pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer,lymphoma, myeloma, and leukemias (e.g., lymphocytic leukemias andmyelogenous leukemias).

In some cases, methods described herein also can include identifying amammal as having cancer. Examples of methods for identifying a mammal ashaving cancer include, without limitation, physical examination,laboratory tests (e.g., blood and/or urine), biopsy, imaging tests(e.g., X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans(e.g., bone scans), endoscopy, and/or genetic tests. Once identified ashaving cancer, an infectious disease, and/or a genetic disease, a mammalcan be administered or instructed to self-administer one or more arecombinant Ads described herein (e.g., recombinant Ads having oncolyticanti-cancer activity such as recombinant Ad657s) or a nucleic acid(e.g., an expression vector) encoding one or more a recombinant Adsdescribed herein (e.g., recombinant Ads having oncolytic anti-canceractivity such as recombinant Ad657s).

One or more recombinant Ads described herein (e.g., recombinant Adshaving oncolytic anti-cancer activity such as recombinant Ad657s) can beadministered by any appropriate route. In some cases, administration canbe local administration. In some cases, administration can be systemicadministration. Examples of routes of administration include, withoutlimitation, intravenous, intramuscular, subcutaneous, oral, intranasal,inhalation, transdermal, parenteral, intratumoral, retro-ureter,sub-capsular, vaginal, and rectal administration. In cases wheremultiple rounds of treatment are administered, a first round oftreatment can include administering one or more recombinant Adsdescribed herein to a mammal (e.g., a human) by a first route (e.g.,intravenously), and a second round of treatment can includeadministering one or more recombinant Ads described herein to a mammal(e.g., a human) by a second route (e.g., intramuscularly).

As used herein, the term “pharmaceutical composition” refers to thecombination of one or more recombinant and/or chimeric Ads of thepresent invention with a carrier, inert or active, making thecomposition especially suitable for therapeutic use. One or morerecombinant Ads described herein (e.g., recombinant Ads having oncolyticanti-cancer activity such as recombinant Ad657s) can be formulated intoa composition (e.g., a pharmaceutical composition) for administration toa mammal (e.g., a mammal having, or at risk of having, cancer). Forexample, one or more recombinant Ads can be formulated into apharmaceutically acceptable composition for administration to a mammalhaving, or at risk of having, cancer. In some cases, one or morerecombinant Ads can be formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Apharmaceutical composition can be formulated for administration in solidor liquid form including, without limitation, sterile solutions,suspensions, sustained-release formulations, tablets, capsules, pills,powders, wafers, and granules. Pharmaceutically acceptable carriers,fillers, and vehicles that may be used in a pharmaceutical compositiondescribed herein include, without limitation, saline (e.g.,phosphate-buffered saline, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-basedsubstances, sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin, 18th Edition. The selectionand use of suitable excipients is taught in Gennaro, ed., Remington: TheScience and Practice of Pharmacy, 20th Ed. (Lippincott Williams &Wilkins 2003).

A composition (e.g., a pharmaceutical composition) including one or morerecombinant Ads described herein (e.g., recombinant Ads having oncolyticanti-cancer activity such as recombinant Ad657s) can be administered toa mammal (e.g., a mammal having, or at risk of having, cancer) as avaccine. A vaccine can be prophylactic or therapeutic.

In some cases, methods described herein also can include administeringto a mammal (e.g., a mammal having cancer) one or more additional agentsused to treat a cancer. The one or more additional agents used to treata cancer can include any appropriate cancer treatment. In some cases, acancer treatment can include surgery. In some cases, a cancer treatmentcan include radiation therapy. In some cases, a cancer treatment caninclude administration of a pharmacotherapy such as a chemotherapy,hormone therapy, targeted therapy, and/or cytotoxic therapy. Forexample, a mammal having cancer can be administered one or more arecombinant Ads described herein (e.g., recombinant Ads having oncolyticanti-cancer activity such as recombinant Ad657 and variants thereof) andadministered one or more additional agents used to treat a cancer. Incases where a mammal having cancer is treated with one or more arecombinant Ads described herein and is treated with one or moreadditional agents used to treat a cancer, an infectious disease, and/ora genetic disease, the additional agents used to treat a cancer, aninfectious disease, and/or a genetic disease can be administered at thesame time or independently. For example, one or more a recombinant Adsdescribed herein and one or more additional agents used to treat acancer, an infectious disease, and/or a genetic disease can beformulated together to form a single composition. In some cases, one ormore a recombinant Ads described herein can be administered first, andthe one or more additional agents used to treat a cancer, an infectiousdisease, and/or a genetic disease administered second, or vice versa.

EXAMPLES Example 1. Adenoviruses

For 50 years, there were only four known species C human species Adsincluding Ad1, Ad2, Ad5, and Ad6 (see, e.g., Weaver et al., 2011Virology. 412:19-27). In 2001, a fifth species C adenovirus wasidentified as field isolate strain #16700, and virus neutralizationtesting with antisera against Ad1, 2, 5, and 6 demonstrated high levelsof neutralization (reciprocal titers of 500-16,000) when each antiserawas used against its cognate virus (Lukashev et al., 2008 J Gen Virol.89:380-388). In contrast, anti-Ad1, 2, and 5 antibodies have weakcross-reactivity against #16700 (reciprocal titers of 32-64). Anti-Ad6sera demonstrated higher cross-reactivity against #16700, butneutralization required 10-fold higher concentrations of sera toneutralize #16700 when compared with Ad6 itself. Subsequent sequencecomparisons confirmed #16700 as a novel species C adenovirus and renamedit as Ad57 (see, e.g., Walsh et al., 2011 J. Clin Microbiol.49:3482-3490).

15 human Ads were evaluated for oncolytic activity against breast,ovarian, liver, prostate, kidney, and B cell malignancies. In testsagainst DU145 human prostate tumors, species C Ad6 was more potent aftersingle intratumoral or intravenous (i.v.) injection than species C Ad5or species B viruses Ad11 and Ad35. Ad6 was also more effective than Ad5and Ad11 in immunocompetent Syrian hamsters.

Construction of Recombinant Adenoviruses.

Recombinant adenoviruses were constructed by recombinant DNA technologyutilizing methods known to those skilled in the art. A recombinant Ad isderived from a first Ad (e.g., can include a genome of a first Ad, suchas Ad6) and may include hexon HVRs from a second Ad such as Ad57. Incases where a recombinant Ad includes an Ad6 genome and Ad57 hexon HVRs,the recombinant Ad can be a chimeric Ad referred to as Ad657.

To obtain Ad657, an Ad57 HVR sequence was synthesized and inserted intothe Ad6 hexon in a plasmid with an FRT-Zeocin®-FRT cassette between pVIand hexon. This was recombined into various pAd6 plasmids to generateAd657 and variants thereof. The amino acid sequence of the Ad657 hexonis set forth in SEQ ID NO:49. See FIGS. 34 and 59-65 for plasmid maps ofAd657 variants.

With respect to variants of Ad657, the Ad57 HVR sequence was synthesizedwith HVR1 modified with a cysteine, flexibility amino acids, andrestriction sites to allow insertions of other peptides. This wasinserted into the Ad6 hexon in a plasmid with an FRT-Zeocin®-FRTcassette between pVI and hexon. This was recombined into various pAd6plasmids to generate Ad657 variants with cysteine in HVR1, the variantreferred to as Ad657-HVR1-XXA comprises the hexon having the amino acidsequence of SEQ ID NO:50.

With respect to variants of Ad657, the Ad57 HVR sequence was synthesizedwith HVR5 modified with a cysteine, flexibility amino acids, andrestriction sites to allow insertions of other peptides. This wasinserted into the Ad6 hexon in a plasmid with an FRT-Zeocin®-FRTcassette between pVI and hexon. This was recombined into various pAd6plasmids to generate Ad657 variants with cysteine in HVR5, the variantreferred to as Ad657-HVR5-XXA comprises the hexon having the amino acidsequence of SEQ ID NO:51.

With respect to variants of Ad657, the Ad57 HVR sequence was synthesizedwith HVR1 modified with a cysteine, flexibility amino acids, andrestriction sites to allow insertions of other peptides. This wasinserted into the Ad6 hexon in a plasmid with an FRT-Zeocin®-FRTcassette between pVI and hexon. This was recombined into various pAd6plasmids to generate Ad657 variants without cysteine in HVR1, but withrestriction sites allowing peptide insertions into HVR1, the variantreferred to as Ad657-HVR1-XA comprises the hexon having the amino acidsequence of SEQ ID NO:52.

With respect to variants of Ad657, the Ad57 HVR sequence was synthesizedwith HVR5 modified with a cysteine, flexibility amino acids, andrestriction sites to allow insertions of other peptides. This wasinserted into the Ad6 hexon in a plasmid with an FRT-Zeocin®-FRTcassette between pVI and hexon. This was recombined into various pAd6plasmids to generate Ad657 variants without cysteine in HVR5, but withrestriction sites allowing peptide insertions into HVR5, the variantreferred to as Ad657-HVR5-XA comprises the hexon having the amino acidsequence of SEQ ID NO:53.

With respect to variants of Ad657, the Ad657 HVR1-XA sequence wasmodified by insertion of a biotin acceptor peptide into HVR1. This wasrecombined into various pAd6 plasmids to generate Ad657 variants a BAPin HVR1, the variant referred to as Ad657-HVR1-PSTCD comprises the hexonhaving the amino acid sequence of SEQ ID NO:54.

The insertion of a biotin acceptor peptide detargets the virus variantsfrom the liver, allows the virus to be retargeted with avidin orstreptavidin and biotinylated ligands, and allows the virus to bepurified on monomeric avidin or streptavidin columns.

With respect to variants of Ad657, the Ad657 HVR1-XA sequence wasmodified by insertion of a biotin acceptor peptide into HVR1. This wasrecombined into various pAd6 plasmids to generate Ad657 variants a BAPin HVR1, the variant referred to as Ad657-HVR5-PSTCD comprises the hexonhaving the amino acid sequence of SEQ ID NO:55.

With respect to variants of Ad657, the Ad657 HVR5-XA sequence wasmodified by insertion of a synthetic V1/V2 loop from HIV envelope intoHVR5, the variant referred to as Ad657-HVR5-V1/V2 comprises the hexonhaving the amino acid sequence of SEQ ID NO:56.

The insertion of a synthetic V1/V2 loop from HIV envelope allows displayof this antigen to serve as a vaccine as well as retargeting by bindingto proteins that interact with HIV envelope.

With respect to variants of Ad657, the Ad657 HVR5-XA sequence wasmodified by insertion of synthetic peptides from human papilloma virusinto HVR5, the variant referred to as Ad657-HVR5-HPV comprises the hexonhaving the amino acid sequence of SEQ ID NO:57.

The insertion of synthetic peptides from human papilloma virus allowsdisplay of HPV peptides as antigens for vaccine purposes as well as forretargeting by binding to proteins that interact with HPV peptides.

In another aspect of the invention, chimeric Ads were generated whichhave an Ad6 HVR1 and Ad57 HVRs 2-7, the chimera, referred to as Ad6/57HVR chimera, comprises the hexon having the amino acid sequence of SEQID NO:58.

In yet another aspect of the invention, chimeric Ads were generatedwhich have Ad6 HVR1 and 7 and Ad57 HVRs 2-6, the chimera, referred to asAd6/57/6 HVR chimera, comprises the hexon having the amino acid sequenceof SEQ ID NO:59.

Example 2. Retargeted and Detargeted Recombinant Adenovirus for GeneDelivery

Adenoviruses are robust vectors for gene delivery and gene-basedimmunization. The archetype adenovirus used for the vast majority ofthese application has been human species C adenovirus serotype 5(HAdV-05 or Ad5). In vitro, Ad5 binds and enters cells through thecombined interactions of its fiber and penton base proteins with cellsurface receptors. The trimeric fiber binds the coxsackie-adenovirusreceptor (CAR), and cells that lack CAR are relatively resistant toinfection unless they also express α_(v) integrins that can be bound byan RGD motif on the penton base.

In vivo, these interactions are still utilized, but their importancevaries by injection route. If injected directly into a solid tissue ortumor, CAR and integrin interactions dominate. If injected intravenously(IV), these interactions become secondary due to the effects of Ad5binding to vitamin-K-dependent blood clotting factors. Blood factor X(FX) binds with subnanomolar affinity to the hexons of Ad5 and,consequently, enables Ad5 to efficiently transduce liver hepatocytesafter IV injection. In the absence of FX, liver transduction isdrastically reduced.

Adenoviral vectors are somewhat unique in their ability to carry verylarge cDNA sequences of up to 36 kilobase pairs (kbp) when compared toother vectors like adeno-associated virus (AAV) vectors with only 4.5 kbof DNA sequence. This payload capacity justified early exploration of Advectors for muscle gene therapy when delivering very large transgeneslike the 14 kbp dystrophin cDNA. IV administration in newborn mice canmediate muscle gene delivery, but this ability is lost in adult mice.The decreased transfection with age is due in part to the very largesize of Ad virions (i.e. 100 nm) as well as the loss of CAR receptor onmuscle cells with aging. The intramuscular (IM) route is by far the mostpopular route for gene-based vaccines when using Ad5 and other serotypesdespite the fact that CAR is absent on skeletal muscle cells.

Therefore, Ad5 and other Ad serotype transduction of muscle can beadequate for gene therapy or gene-based vaccination. However, theabsence of the virus' primary receptor in the muscle reduces theefficacy of the virus and requires more vector to be delivered toachieve desired effects.

Construction of Peptide-Modified Hexons in Adenovirus.

12 amino acid (12-mer) peptides on C2C12 mouse muscle cells wereselected from a random peptide library displayed between the H and I βsheets from the knob region of Ad5 (FIG. 1A). Peptides 12.51(TARGEHKEEELI; SEQ ID NO:1) and 12.52 (LRQTGAASAVWG; SEQ ID NO:2) wereselected against myoblasts with pre-clearing against non-target cells toobtain peptides which would be specific for binding muscle cells. Inmost cases, small peptides have relatively low affinity. It wastherefore reasoned that displaying these muscle-selected peptides on the720 copies of the Ad5 hexon might enable better muscle gene delivery.This insertion site might also have the benefit of inactivating FXbinding to the hexon to “detarget” the vector from the liver if anyvector leaked into the blood after IM injection.

Inserting these muscle-selected peptides between two other β sheetswhich also constrains a hypervariable loop on the virus, the ability ofthe modified Ads to modulate tropism was evaluated. Peptides 12.51 and12.52 were introduced into the hypervariable region (HVR) 5 loopconstrained by the β7 and β8 sheets in Ad5 hexon. The in vivo ability ofthese viruses to transduce tissues after intravenous and intramuscularinjections in mice and in hamsters was evaluated.

Adenoviruses

E1-deleted Ad5-GL (RD-Ad5-GL) expresses a green fluorescentprotein-luciferase (GFP-Luciferase, GL) fusion protein as describedelsewhere (see. e.g., Crosby et al., 2002 J. Virol., 76:6893-6899; Khareet al., 2011 Mol. Ther., 19:1254-1262; and Khare et al., 2012 J. Virol.,86:2293-2301). Muscle selected peptides 12.51 and 12.52 were inserted inplace of HVRS on Ad5 hexon between its β7 and β8 sheets whichstructurally similar to the H and I β sheets from the knob region of Ad5(FIG. 1A) according to methods known to those skilled in the art. Thepeptides with a flexibility leader replaced the entire HVRS loop in Ad5(FIG. 1B). These modified hexon sequences were introduced intoreplication-defective Ad5 expressing a green fluorescentprotein-luciferase (RD-Ad5-GL) fusion protein by red recombination inbacteria (see, e.g., Campos et al., 2004 Hum. Gene Ther., 15:1125-1130).Peptide modified Ads were constructed by insertion of annealedoligonucleotides encoding the peptides 12.51 and 12.52 (FIG. 1B) intothe plasmid pHVR5 display (FIG. 1A bottom) to yield recombinant Ads,Ad5-HVRS-12.51 and Ad5-HVRS-12.52.

The resulting plasmids were digested and used for red recombination intopAd5-GL. These vectors were rescued in 293 cells, purified on twoconsecutive CsCl gradients and were desalted on Econopac 10-DGchromatography columns (Bio-Rad) into 50 mM Tris pH 8 with 0.5 M sucroseand stored at −80° C.

In Vitro Virus Testing

C2C12 mouse myoblasts were purchased from American Type Tissue Culture(Manassas, VA). 293 cells were obtained from Microbix, Toronto, Ontario,Canada. Cells were maintained in DMEM with 10% FBS Invitrogen.

C2C12 muscle cells were plated in 6 well plates (Corning) the day beforeinfection. Viruses were used to infect cells in DMEM with 5% FBS. Thecells were incubated for 2 days prior to observation under greenfluorescence.

Animal experiments were performed with approval by the Mayo ClinicInstitutional Animal Care and Use Committee under the provisions of theAnimal Welfare Act, PHS Animal Welfare Policy and principles of the NIHGuide for the Care and Use of Laboratory Animals. Female CD-1 mice(Charles River) were anesthetized and injected intramuscularly (IM) orintravenously (IV) with 10¹⁰ vp of the indicated viruses at indicatedtimes. The animals were anesthetized and injected with 3 mg ofd-luciferin (Molecular Imaging Products) and were imaged on a Xenogenimaging system. At later times, the animals were anesthetized and bloodwas collected in serum separators for ELISA.

ELISA was performed as follows. Immulon 4 HBX plates (Thermo) wereincubated with 100 ng per well of GFP protein in 1× phosphate-bufferedsaline (PBS) at 4° C. overnight, washed, and blocked with 5% milk inTRIS-buffered saline with 0.1% Tween 20 (TB ST) at room temperature for2 hours. 1:100,000 to 1:1,1000 dilutions of each serum sample wereprepared in blocking buffer. Wells were washed and 100 μL of each wereadded to GFP-coated plates in triplicate and incubated for 3 hours atroom temperature. Wells were washed and a 1/10,000 dilution of goatanti-mouse-HRP secondary antibody (Pierce Chemical) was added to eachwell. Plates were incubated for 2 hours at room temperature, washed, and50 μL of 1 Step Ultra TMB ELISA (Thermo Fisher Scientific Inc.) wasadded for HRP detection followed by 50 μL of 2 M H₂SO₄. Absorbance at450 nm was determined with a Beckman Coulter DTX 880.

Statistical analyses were performed with Prism (Graphpad). Statisticalsignificance was calculated by one-way ANOVA followed by Tukey's HSD

In Vitro Transduction in Mouse C2C12 Muscle Cells

RD-Ad5-GL, RD-Ad5-GL-HVR5-12.51, and RD-Ad5-HVR5-12.52 were used toinfect mouse C2C12 myoblast cells at varied multiplicities of infection(MOI) in terms of virus particles (vp)/cell. When green fluorescencefrom the GFP fusion protein was observed by fluorescence microscopy,both of the peptide-modified vectors mediated significantly bettertransduction than RD-Ad5-GL (FIG. 2A). When luciferase activity wasmeasured, significant increases were observed in RD-Ad5-GL-12.51 and12.52 infected cells (FIG. 2B). When one of the peptides, 12.51, wasinserted back into the knob region of Ad5, the peptide increased invitro transduction 14-fold on C2C12 myoblasts.

In Vivo Transduction after Intramuscular Injection in Mice

10⁹ vp of Ad5-GL, Ad5-GL-HVR5-12.51, and Ad5-GL-HVR5-12.52 were injectedby the IM route into both quadriceps muscles in mice and luciferaseimaging was performed at varied times (FIG. 3A top). Ad5-GL-HVR5-12.51produced 2 to 3-fold higher luciferase activity than Ad5-GL at all thetime points (p<0.05 at day 1 by one-way ANOVA) (FIG. 3B left).Ad5-GL-HVR5-12.52 activity in the muscle was lower than both Ad5-GL andHVR5-12.51 in contrast to its stronger activity in vitro.

In Vivo Transduction after Intravenous Injection in Mice

3×10¹⁰ vp of Ad5-GL, Ad5-GL-HVR5-12.51, and Ad5-GL-HVR5-12.52 wereinjected by the IV route in mice and luciferase imaging was performed(FIG. 3A bottom). In contrast to the results in the muscle, onlyunmodified Ad5-GL mediated strong liver transduction. Liver transductionby Ad5-GL was 60-fold higher than both Ad5-GL-HVRS-12.51 andAd5-GL-HVR5-12.52 (p<0.001 at day 1 by one-way ANOVA) (FIG. 3B right)demonstrating that the recombinant Ads target muscle tissue whiledecreasing off target infection in the liver.

In Vivo Transduction after Intramuscular Injection in Hamsters

To test if the 12.51 modified vector works in other species than mice,10¹⁰ vp of Ad5-GL and Ad5-GL-HVR5-12.51 were injected IM into bothquadriceps of larger Syrian hamsters and luciferase imaging wasperformed 24 hours later (FIG. 4 ). In this case, Ad5-GL-HVR5-12.51mediated 7-fold higher luciferase activity than Ad5-GL (p<0.01 at day 1by one-way ANOVA).

Gene-Based Immunization after Intramuscular Injection in Mice

16 weeks after IM injection, sera were collected the mice treated asdescribed above and as shown in FIG. 3 and analyzed in serial dilutionsby ELISA for antibodies against transgene-encoded GFP (FIG. 5 ). AllAd-injected mice generated significant anti-GFP antibodies when comparedto the PBS group at sera dilutions of 1:10,000 to 1:1000 (p<0.0001 byone-way ANOVA). However, Ad5-GL-HVR-12.51 produced higher antibodiesthan either Ad5 or Ad5-HVR5-12.52. At 1:1000 to 1:10,000 dilutions ofsera, Ad5-GL-HVR-12.51 was significantly higher than Ad5-GL (p<0.01 to0.0001 by one-way ANOVA). At a 1:1000 dilution of sera, 12.51 wassignificantly higher than 12.52 (p<0.05). Ad5-GL-12.52 was significantlyhigher than Ad5-GL at 1:1000 and 1:10,000 dilutions of sera (p<0.05 to0.001).

This example demonstrates that peptides selected in a compatiblestructural context on phage libraries can be translated into the Adhexon protein. For example, for the 12.51 peptide, this insertion siteincreases muscle transduction while decreasing off target infection inthe liver. Thus, such a recombinant Ad which targets muscle tissue maybe used as a vector for gene-based muscle vaccination or for genetherapy application/delivery to the muscle.

A further aspect of the invention relates to recombinant and/or chimericAds which comprise other cell targeting peptides inserted into Ad657HVRs and into Ad6 and C68 HI loops are described in Table 1.

TABLE 1Other Cell Targeting Peptides Inserted into Ad657 HVRs and into Ad6 and C68 HI LoopsVSV cell binding peptide GTWLNPGFPPQSCGYATVT (SEQ ID NO: 4)RGD-4C integrin binding peptide CDCRGDCFC (SEQ ID NO: 5)12.51 phage-selected peptide TARGEHKEEELI (SEQ ID NO: 1)12.52 phage-selected peptide LRQTGAASAVWG (SEQ ID NO: 2)12.53 phage-selected peptide ARRADTQWRGLE (SEQ ID NO: 3)alpha4 binding peptide NMSLDVNRKA (SEQ ID NO: 6)L10.1 lung binding peptide WTMGLDQLRDSSWAHGGFSA (SEQ ID NO: 9)L10.2 lung binding peptide RSVSGTEWVPMNEQHRGAIW (SEQ ID NO: 10)L10.5 lung binding peptide TELRTHTSKELTIRTAASSD (SEQ ID NO: 11)S5.1 muscle binding peptide DRAIGWQDKLYKLPLGSIHN (SEQ ID NO: 12)DU9C.1 prostate cancer binding peptideMGSWEKAALWNRVSASSGGA (SEQ ID NO: 13)DU9C.2 prostate cancer binding peptideMAMGGKPERPADSDNVQVRG (SEQ ID NO: 14)DU9A.7 prostate cancer binding peptideMASRGDAGEGSTQSNTNVPS (SEQ ID NO: 15) XS.1 dendritic cell binding peptideGPEDTSRAPENQQKTFHRRW (SEQ ID NO: 17)REDV endothelial cell binding peptide REDVY (SEQ ID NO: 46)SKBR5C1 breast cancer cell binding peptideGQIPITEPELCCVPWTEAFY (SEQ ID NO: 20)231R10.1 breast cancer cell binding peptidePQPPNSTAHPNPHKAPPNTT (SEQ ID NO: 21)HepaCD8 hepatocellular cancer binding peptideVRWFPGGEWGVTHPESLPPP (SEQ ID NO: 22)HI Met 231 3-4 breast cancer binding peptideISLSSHRATWVV (SEQ ID NO: 47) B Cell Cancer Selected Peptides: 1-1GVSKRGLQCHDFISCSGVPW (SEQ ID NO: 29) 1-2NQSIPKVAGDSKVFCWWCAL (SEQ ID NO: 30) 1-3QSTPPTKHLTIPRHLRNTLI (SEQ ID NO: 31) 1-4DMSFQLVTPFLKALPTGWRG (SEQ ID NO: 32) 1-5GGHGRVLWPDGWFSLVGISP (SEQ ID NO: 33) 1-6QIMMGPSLGYYMPSESIFAY (SEQ ID NO: 35) 2-11ISWDIWRWWYTSEDRDAGSA (SEQ ID NO: 36) 2-14VWGMTTSDHQRKTERLDSPE (SEQ ID NO: 37) 2-20MTSAQTSEKLKAETDRHTAE (SEQ ID NO: 38) 2-9MGSRSAVGDFESAEGSRRP (SEQ ID NO: 39) 3b-6MGRTVQSGDGTPAQTQPSVN (SEQ ID NO: 40) 4*-5MARTVTANVPGMGEGMVVVP (SEQ ID NO: 41) Small BAP biotin acceptor peptideGLNDIFEAQKIEWH (SEQ ID NO: 24) calmodulin binding peptideCAAARWKKAFIAVSAANRFKKIS (SEQ ID NO: 25)

DNA encoding the indicated peptides and its complementary DNA wassynthesized flanked by cohesive ends for ligation into Ad plasmids, forexample, XA hexon plasmids or pAd6-NdePfl fiber shuttle plasmids. Theseannealed oligonucleotides were ligated into HVRs or the HI loop of Ads.These plasmids were used to recombine into various Ad backbone plasmids.

Some peptides serve to target novel receptors on cells. Others like thesmall BAP can be used for avidin targeting and purification if the virusis grown in cells expressing bacterial biotin ligase BirA. Thecalmodulin peptide allows the virus to bind to calmodulin orcalmodulin-fusion proteins for retargeting or for virus purification.

Thus, such a recombinant Ads which targets specific tissues/cellreceptors may be used as a vector for gene-based vaccination or for genetherapy application in the targeted cells and/or tissues.

Example 3. Insertion of Individual HVRs from Different Ad Serotypes withthe Insertion of Cell Targeting/Detargeting Peptides or Novel AminoAcids

Hexon shuttle plasmid maps (FIG. 34 ) show the combination of theinsertion of individual HVRs from different Ad serotypes with theinsertion of cell targeting/detargeting peptides or novel amino acidssuch as cysteine into the hexon for targeted chemical modification andshielding.

In certain embodiments, cell binding peptides 12.51, VSV, RGD (seeTable 1) are inserted into HVR 1 or HVR 5, which embodiments serve asexamples of inserting these and other peptides in any of the HVRs of anAd (FIG. 34 ). Another example shows insertion of a biotin acceptorpeptide (BAP) is inserted into these HVRs allowing for vectorretargeting with avidin or streptavidin and biotinylated ligands or withavidin- or streptavidin fusion proteins. BAP insertion also allows theviruses to be purified on monomeric avidin or streptavidin columns forvector production. Likewise, Ad57-HVR1-XXA and XA shows the example ofinserting a cysteine into this site to allow targeted chemicalmodification with maleimide or other cysteine-reactive agents (FIG. 34).

These embodiments have been applied also in the context of Ads whichcombine different HVRs from different Ads (i.e., shuffling HVRs). Forexample, HVR1 of Ad6 with HVRs 2-7 of Ad57 or HVR1 and 7 of Ad6 withHVRs 2-6 of Ad57. In a further embodiment, a 6/57/6 virus has HVRs 1 and7 from Ad6 and HVRs 2-6 from Ad657.

Example 4. Targeted Chemical Conjugation of Cysteine-ModifiedHexon-Modified Ad657-HVR5C

FIG. 35 is a depiction of Ad variants showing the combination ofinsertion of individual HVRs from different Ad serotypes with theinsertion of novel amino acids such as cysteine into the hexon fortargeted chemical modification and shielding.

Comparison of the effects of non-targeted chemical conjugation totargeted chemical conjugation on shielding and function ofcysteine-modified hexon-modified Ad657-HVR1C (FIG. 36 ). This exampledemonstrates the ability to target polymer and other chemicalmodifications to cysteines inserted into an Ad hexon. Untargeted PEGinactivates virus infection whereas cysteine-targeting PEGylationretains virus functions.

In an aspect of the invention, the use of polymers or insertedpeptides/proteins to detarget, retarget, and shield from antibodies,proteins, cells is contemplated. FIG. 54 depicts sites of Ad HVRs whichmay be modified, for example, by PEGylation or “BAPylation”.

In an embodiment, the different Ad serotypes and/or variants comprisepolymer shielding to allow multi dosing of Ad6 and Ad657 variants. Anexemplary therapeutic cycle where Ad6 and Ad657 can be used for multiplerounds of treatment by serotype-switching in combination with covalentpolymer conjugation is shown (FIG. 41B).

Ad657-HVR1C expressing GFPLuciferase was produced from a cells andpurified on CsCl gradients. The virus was covalently modified with 5 kDapolyethylene glycol (PEG). The virus was treated with either NHS-PEGthat reacts randomly with amines/lysines on viral proteins or withmaleimide PEG that reacts specifically with cysteine that was insertedinto HVR1 using the XXA shuttle plasmid. These unmodified or modifiedviruses were then purified by a final CsCl spin followed by desalting.The indicated virus were separated on SDS-PAGE gels, stained withSyproRuby, and visualized by imaging (FIG. 36A). This shows thatNHS-PEGylation randomly modifies many viral proteins as demonstrated byincreases in the apparent mass of the proteins (indicated by arrows). Incontrast, targeted maleimide PEG reaction with the cysteine in HVR1modifies only hexon and does not damage other viral capsomer proteins.The effects of PEGylation on virus function was evaluated.

The indicated viruses were incubated with A549 cells and their abilityto infect the cells was measured by luciferase assay. This shows thatrandom NHS-PEGylation reduces virus activity more than 90% whereasmaleimide-PEG does not (FIG. 36B).

Immune competent Syrian hamsters were engrafted with subcutaneous HaKkidney cancer tumors. When these reached 200 μl volume, they wereinjected a single time by the intravenous route with the indicated Ad6viruses constructed with and without E3 (DE3) and with or without randomNHS-PEGylation. Tumor sizes were measured over time. The data shows thatdeleting all E3 genes in the oncolytic virus Ad6-deltaE3-Luc makes thevirus less effective at reducing tumor volume than the oncolytic parentvirus, Ad6-Luc. The data also shows that Ad6 can be PEGylated and retainefficacy (see Ad6-Luc vs. Ad6-Luc-20 kDa PEG) (FIG. 58 ).

Targeted chemical conjugation of cysteine-modified hexon-modified Ads,for example Ad657-HVR5C. Ad657-HVR5C expressing GFPLuciferase wasproduced from cells and purified on CsCl gradients. The virus wascovalently modified with maleimide-IR800 near-infrared fluorophore,maleimide-biotin, or 5 kDa maleimide-PEG that reacts specifically withcysteine that was inserted into HVR5 using its XXA shuttle plasmid. Theindicated Ads and modified Ads were separated on SDS-PAGE gels, stainedwith SyproRuby, and visualized by imaging (FIG. 37A). SDS-PAGE of viralproteins followed by near infrared imaging demonstrates that the HVR-Ccan be tagged with an imaging agent (FIG. 37B). The effects ofPEGylation on in vivo Ad virus function was demonstrated by injectingPEGylated Ad virus intraperitoneally. The ability to infect cells intumor bearing mice is demonstrated by detectable luciferase activity byimaging. FIG. 37C demonstrates the ability to target polymer and otherchemical modifications to cysteines inserted into the Ad657 hexonregion. What is more, it is demonstrated that PEGylation de-targetsadenovirus to liver in vivo (FIG. 50 ).

Example 5. Expression of Human Granulocyte-Macrophage Colony StimulatingFactor (GMCSF) by Ad657

Ad657 carrying the cDNA for human GMCSF was used to infect A549 cellsand varied amounts of the supernatant were added to GMCSFgrowth-dependent TF-1 cells. Increased cell number indicates expressionof the functional human cytokine (FIG. 27 ). The data demonstrates thatrecombinant Ads may be utilized for expression of heterologous proteins.

Example 6. Oncolytic Adenovirus Ad657 for Systemic Virotherapy AgainstCancer Cells

An alignment of selected full Ad genomes produces a phylogenetic treethat clusters Ad57 with other species C viruses with most homology withAd6 is shown in FIG. 6 .

Ad57 appears nearly identical to Ad6 with sequence divergence in hexonhypervariable regions (HVRs) and in E3 immune evasion genes (FIG. 7 ).Other exposed viral capsid proteins including fiber, penton base, Ma,and IX are virtually identical between Ad6 and Ad57. The neutralizationdata are consistent with the fact that most adenovirus-neutralizingantibodies target the HVRs on Ads. The low cross-reactivity between Ad6antisera and Ad57 is thought to be due to antibodies that may targettheir common fiber protein (Lukashev et al., 2008 J Gen Virol.89:380-388).

In this example, the utility of Ad657 as an oncolytic against humanprostate cancer is demonstrated. The Ad6 HVRs were replaced with thosefrom Ad57 to generate a chimeric species C oncolytic virus called Ad657.Ad657 and Ad6 were tested as systemic oncolytic therapies by single i.v.injection in nude mice bearing human prostate cancer tumors. The liverand tumor tropism of this virus were evaluated in mouse models ofprostate cancer as follows.

DU145 human prostate carcinoma cells were purchased from American TypeCulture Collection (ATCC; Manassas, VA, USA) and verified to be specificpathogen free by IMPACT testing by RADIL. 293 cells were obtained fromMicrobix, Toronto, Ontario, Canada. Cells were maintained in DMEM with10% FBS (Invitrogen, Grand Island, NY, USA).

The genome of Ad6, Tonsil 99 strain (ATCC VR-1083), was cloned asdescribed elsewhere (see, e.g., Weaver et al., 2013 PLoS One. 8:e73313).A cassette corresponding to the Ad57 hexon between a natural ApaI andSacI sites was synthesized by Genscript. This fragment was cloned intothe shuttle plasmid pUC57-Ad6 Hexon-FZF containing the Ad6 pVI and hexongenes with a FRT-Zeocin® resistance gene-FRT cassette between them forhomologous recombination in bacteria as described elsewhere (see, e.g.,Campos et al., 2004 Hum Gene Ther. 15:1125-1130; and Khare et al., 2012J Virol. 86:2293-2301). The Ad6 ApaI-SacI fragment was replaced with theAd57 fragment generating the plasmid pUC57-Ad6/57 Hexon-FZF. This wasrecombined into the Ad6 genome by red recombination (Campos et al., 2004Hum Gene Ther. 15:1125-1130). FIG. 59 shows a plasmid map of Ad657 withE3 deletion. Viruses were rescued by transfection into 293 cells andproduced from a 10 plate CellStack (Corning Life Sciences, Lowell, MA,USA). Viruses expressing a green fluorescent protein-luciferase(GFP-Luc) fusion protein have a CMV-GFP-Luc expression cassette insertedbetween the Ad fiber and E4 and an E3 deletion to make space for thisinsertion. Viruses were purified on two CsCl gradients, and viralparticle (vp) numbers were calculated by OD260.

To examine in vitro oncolytic activity, cells were treated at theindicated multiplicities of infection (MOI) in terms of vp/cell in DMEMwith 5% FBS and antibiotic-antimycotic (Invitrogen, Grand Island, NY,USA). Five days later, media was removed and the cells were treated withcrystal violet (0.05% crystal violet, 3.7% formaldehyde, inphosphate-buffered saline; Invitrogen, Grand Island, NY, USA) for 10minutes. The cells were washed twice with PBS and then incubatedovernight at 37° C. in 0.1% sodium dodecyl sulfate in PBS to solubilizethe crystal violet. Crystal violet absorbance was measured at OD595 on aBeckman Coulter DTX 880 plate reader. Cell viability (%) was calculatedby dividing the OD of the samples by the mean OD of untreated controlcells on the same 96-well plate and multiplying this number by 100.

Animals were housed in the Mayo Clinic Animal Facility under Associationfor Assessment and Accreditation of Laboratory Animal Care guidelines.The studies were approved by the Mayo Clinic Animal Use and CareCommittee under the provisions of the Animal Welfare Act, PHS AnimalWelfare Policy. Subcutaneous tumors were initiated in 4-week-old nudemice (Harlan Sprague Dawley, Indianapolis, IN, USA) by injectingsubcutaneously (s.c.) with 1×10⁷ DU145 cells in 100 μL of DMEM/50%Matrigel (BD Biosciences, San Jose, CA, USA). Tumor volumes werecalculated using the equation width²×length×½. When tumors reached ˜200μL in volume, mice were distributed into different groups and weretreated by a single i.v. injection tail vein. Animals were euthanizedwhen the tumor volume reached 2000 μL or if animals were moribund, indistress, or if the skin ruptured over the tumor.

For blood alanine aminotransferase (ALT) measurements, groups of sixC57BL/6 mice were injected i.v. with 10¹¹ vp of Ad5, Ad6, or Ad657 bytail vein and blood was collected 3 days later for ALT measurement usingALT Activity Assay (Sigma-Aldrich, St. Louis, MO, USA).

Statistical analysis was performed with Prism (Graphpad) by repeatedmeasures ANOVA or one-way ANOVA followed by Tukey's HSD test.Kaplan-Meier survival curves were plotted and compared by log rank test.

The capsomer genes of Ad57 are nearly identical to Ad6 with theexception of their hexon HVRs (FIGS. 6 and 7 ). To generate a chimericvirus of Ad57 and Ad6, a cassette corresponding to the Ad57 hexon HVRswas recombined into the wild-type Ad6 genome (FIG. 8 ). This virus wasrescued and produced in 293 cells and purified on CsCl gradients. Giventhat the base viral genome is Ad6, these hexon chimeric viruses arereferred to as Ad657 (FIG. 59 ).

In vitro oncolytic activity was evaluated by infecting LNCap and DU145cells with 10, 100 or 1000 vp/cell. To compare liver damage by Ad5, Ad6,and Ad657, a high dose of 10¹¹ vp of each virus were injected by tailvein into immunocompetent C57BL/6 mice. Ad5-injected animals becamemoribund within 2 days and had to be euthanized (FIG. 10A). Survival forAd5 and Ad6 was significantly lower when compared with PBS (p=0.0001 and0.0009, respectively, by log-rank analysis). Survival for Ad657 was alsoreduced when compared with PBS (p=0.0578). Survival after exposure toAd6 or Ad657 was significantly better than in Ad5-treated mice (p=0.0001and 0.0001, respectively). Ad6 and Ad657 survival were not statisticallydifferent (p=0.248).

ALT was measured in the blood 3 days after injection in surviving Ad6and Ad657 animals. Ad5-treated animals were not tested, since most ofthe group needed to be sacrificed. This assay showed that Ad6 provokedrelatively low levels of liver damage in terms of liver ALT enzymerelease in the blood (FIG. 10B). Both Ad6 and Ad657 groups had low, butsignificant, ALT levels when compared with PBS-treated mice (p<0.001 byone-way ANOVA with Tukey's multiple comparison test for both viruses).Ad657 had lower ALT levels than Ad6 (p<0.001 by ANOVA). This isconsistent with higher levels of Ad6 infection in the liver than Ad657after i.v. injection of luciferase expressing viruses (FIG. 12 ). OneAd657 animal was lost following bleeding on day 3. By 6 days, most ofthe Ad6 animals became moribund (FIG. 10A). In contrast, 50% of Ad657animals survived beyond 2 weeks of the treatment.

To compare the oncolytic activity of Ad6 and Ad657 against human DU145prostate tumors, nude mice were engrafted s.c. with DU145 cells. Animalswere distributed into groups with similar tumor sizes averaging 200 μLand groups of nine mice were treated a single time by the i.v. routewith a dose of 3×10¹⁰ vp of Ad6 or Ad657 (FIG. 11 ).

This single i.v. injection of Ad6 and Ad657 reduced tumor sizes whencompared with PBS-injected control animals. Tumors were significantlysmaller in the Ad6 group within 7 days when compared with the PBS group(p<0.05 by two-way ANOVA with Tukey's multiple comparison test). Tumorsin the Ad657 group were significantly different from those in the PBSgroup by day 14 (p<0.01 by ANOVA). Both Ad6 and Ad657 maintainedsignificant differences with PBS through day 38 (p<0.0001 by two-wayANOVA). This comparison ended on day 38 when the first animal in the PBSgroup had to be sacrificed since later comparison would be skewed due tothe change in animal numbers. Tumor sizes in the Ad6 and Ad657 groupswere not significantly different until day 38, when Ad657 had asignificantly higher tumor volume (p<0.05) by two-way ANOVA (FIG. 11A).This difference between Ad6 and Ad657 tumor sizes persisted until day 52(p=0.04 by T-test), and then the tumors were not significantly differentafter this time.

When survival due to all causes was assessed, both Ad6 and Ad657significantly extended survival when compared with PBS-treated animals(FIG. 11B, p<0.01 and 0.05, respectively, by log-rank analysis). Ad6survival due to all causes was significantly better than Ad657 (p<0.05).However, this was an artifact of survival attributed to all becausethree of the Ad657 animals had to be sacrificed per Institutional AnimalCare and Use Committee (IACUC) guidelines due to the formation of ulcerson the skin over the tumor rather than due to excess tumor size. In somecases, ulceration is actually associated with effective tumor control.Like Ad6, Ad657 expressing GFP-luciferase produced significantluciferase activity in distant DU145 subcutaneous tumors after a singlei.v. injection (FIG. 12 and FIG. 13 ). This suggests that both Ad6 andAd657 can mediate oncolytic effects in prostate tumors after a singlesystemic treatment.

This example demonstrates that Ad657 may be used as a local or systemiconcolytic virotherapy for prostate cancers. These data also demonstratesurprising effects of serotype-switching with oncolytic species C Ads.

The oncolytic activity of Ads was evaluated in tumor cells and/orcancerous tumors. Ad6 single IV injection vs. A549 lung tumor cells wasevaluated (FIG. 28 ); Ad6 single IV or intratumoral (IT) injection vs.Panc1 pancreatic tumors was evaluated (FIG. 29 ), and Ad6 single IVinjection vs. kidney cancer in immune competent hamsters was evaluated(FIG. 30 ).

FIG. 38 shows luciferase imaging of nude mice. A) 1, 4, 7, 14, 28, and42 days after single I.V. injection of Ad6 treatment vs. distant DU145prostate tumors. B) 3, 7, and 19 days after I.V. injection ofreplicating Ad5-GFPLUC into mice bearing LNCaP prostate tumors.

It may be concluded that Ad6 gets to distant target cells after IVinjection.

In another embodiment, Ads expressing luciferase with and withoutpeptide library generated peptides 12.51 and 12.52 inserted in HVRS ofhexon were incubated on indicated cell lines, B16 melanoma and A549 lungcarcinoma cells, with the indicated numbers of virus particles (vp) andluciferase activity was measured. Improved infection of cancer cells byAds bearing peptide-modified hexons is demonstrated (FIG. 31 ).

Improved Infection of Cancer Cells by Ads Bearing Peptide-ModifiedHexons.

Ads expressing luciferase with and without peptide library generatedpeptides 12.51 and 12.52 inserted in HVR5 of hexon were incubated onindicated hepatocellular carcinoma cell lines with 10⁴ vp of each virusand luciferase activity was measured. Improved infection of cancer cellsby Ads bearing peptide-modified hexons is demonstrated (FIG. 32 ).

Example 7. Divergent HIV-1 Directed Immune Responses Generated bySystemic and Mucosal Immunization with Replicating Single-CycleAdenoviruses in Rhesus Macaques

Most gene-based adenovirus vaccines are replication-defective Ad (RD-Ad)vectors that have their E1 gene deleted to prevent them from replicatingand causing Ad infections. Helper-dependent adenoviruses (HD-Ads) haveall Ad genes deleted and are also replication-defective. An E1-deletedAd vaccine can infect a cell, deliver its one copy of an antigen gene,and express a single copy (e.g., “1×”) of this antigen. They are safe,but do not replicate transgenes or their expression.

In contrast, an E1+ replication-competent Ad (RC-Ad) vaccine can infectthe same cell, replicate the antigen gene DNA 10,000-fold, producesubstantially more antigen, and provoke stronger immune responses thanE1-deleted vectors. While RC-Ad is more potent than RD-Ad,replication-competent Ads can run the real risk of causing frankadenovirus infections in humans.

To take advantage of transgene DNA replication, but avoid the risk ofadenovirus infections, single-cycle Ad (SC-Ad) vectors with a deletionof a gene for a key viral late protein, pIIIa, were developed (Crosby etal., 2014. Virology 462-463:158-165; Crosby et al., 2015 J Virol89:669-675; Anguiano-Zarate et al., 2018 J Infectious Dis 218:1883-1889;and Crosby et al., 2017 Genes (Basel) 8:E79). SC-Ads retain their E1genes to allow it to replicate its genome, but the absence of pIIIablocks the production of infectious progeny viruses. SC-Ads replicatetheir genomes and transgenes as well as RC-Ad (up to 10,000-fold; Crosbyet al., 2014. Virology 462-463:158-165). RC- and SC-Ad produce moretransgene protein than RD-Ad vectors (Crosby et al., 2014. Virology462-463:158-165). SC-Ads generate more robust and more persistent immuneresponses than either RD-Ad or RC-Ads (Crosby et al., 2015 J Virol89:669-675). In head-to-head comparisons, SC-Ad produces significantlyhigher antibodies and better protection against influenza virus (Crosbyet al., 2017 J Virol 91:e00720-16).

In this study, rhesus macaques were immunized with SC-Ads expressingclade B envelope sequences that were obtained from an HIV-1 patientbefore and after their antibody response underwent an expansionneutralization breadth. Macaques were immunized by a single systemic IMimmunization or by single mucosal intranasal (IN) immunization. Theanimals were then boosted by the same or alternative routes with SC-6 Adfollowed by protein boost. This example describes how these variousSC-Ad immunization strategies affected the generation of HIV binding,antibody-dependent cellular cytotoxicity (ADCC) and neutralizingantibodies as well as their effects on cellular immune responsesincluding T follicular helper (pTfh) cells in blood and lymph nodes.

Single-Cycle Adenovirus Expressing HIV-1 Envelope Protein Gp140.

Clade B gp160 envelope sequences that arose before (G4) and immediatelypreceding a peak in the expansion of antibody neutralization breadth(F8) from HIV patient VC10014 (Malherbe et al., 2014 J Virol88:12949-12967) were used as immunogens. Motif-optimized G4 and F8 gp160sequences were recombined into SC-Ads based on human Ad serotypes 6 and57 (see, e.g., Crosby et al., 2014. Virology 462-463:158-165; Crosby etal., 2015 J Virol 89:669-675; Anguiano-Zarate et al., 2018 J InfectiousDis 218:1883-1889; and Nguyen et al., 2018 Oncolytic Virotherapy7:43-51). A control SC-Ad expressing Ebola glycoprotein was also used.Viruses were rescued and purified as described elsewhere (see, e.g.,Crosby et al., 2014. Virology 462-463:158-165; Crosby et al., 2015 JVirol 89:669-675; and Anguiano-Zarate et al., 2018 J Infectious Dis218:1883-1889).

The envelope gene F8 cloned from an HIV Clade B-infected subject used inthe SC-Ad vector was motif-optimized and modified by site-directedmutagenesis to express uncleaved, trimeric gp140. Details of expression,purification, and antigenic characterization have been describedelsewhere (see, e.g., Malherbe et al., 2014 J Virol 88:12949-12967).

Female adult rhesus macaques (Macaca mulatta) of Indian origin weremaintained at the Michael Keeling Center for Comparative Medicine andResearch at the University of Texas MD Anderson Cancer Center, BastropTX in the specific pathogen-free breeding colony. All animal handlingwas carried out in accordance with the policies and procedures of theMayo Clinic and the University of Texas MD Anderson Cancer Center, theprovisions of the Animal Welfare Act, PHS Animal Welfare Policy, theprinciples of the NIH Guide for the Care and Use of Laboratory Animals.

Macaques were anesthetized with ketamine and immunized by the intranasal(IN) or intramuscular (IM) route with 2×10¹⁰ virus particles (vp) of theindicated SC-Ad vaccine. Animals were boosted with 50 μg of purified,trimeric recombinant F8 gp140 combined with Adjuplex™ adjuvant by the IMroute as described elsewhere (see, e.g., Malherbe et al., 2014 J Virol88:12949-12967; and Hessell et al., 2016 J Immunol 196:3064-3078).

Peripheral venous blood samples were collected in EDTA. Before isolationof peripheral blood mononuclear cells (PBMC), plasma was separated andstored immediately at −80° C. PBMCs were prepared from the blood onFicoll-Hypaque density-gradients. Saliva and vaginal swabs collectedwith Wek-Cel Spears in 1 ml of PBS containing protease inhibitors,vortexed, and supernatants were collected after a 2,000 r.p.m.centrifugation as described elsewhere (see, e.g., Kozlowski et al., 1997Infect Immun 65:1387-1394). Samples were kept frozen at −80C untilfurther use.

ELISPOT Assay for Detecting Antigen-Specific IFN-γ Producing Cells

Freshly-isolated PBMCs were stimulated with either F8 gp140 protein (1μg/mL) or heat inactivated Ad6 (7.0×10⁸ vp/well) to determine thenumbers of IFN-γ-producing cells by the Enzyme Linked Immuno Spot(ELISPOT) assay using the methodology described elsewhere (see, e.g.,Nehete et al., 2017 Comp Med 67:67-78; Nehete et al., 2013 PLoS One8:e79836; and Nehete et al., 2017 J Am Assoc Lab Anim Sci 56:509-519).Briefly, aliquots of PBMCs (10⁵/well) were seeded in duplicate wells of96-well plates (polyvinylidene difluoride backed plates, MAIP S 45,Millipore, Bedford, MA) pre-coated with the primary IFN-γ antibody andthe lymphocytes were stimulated with either Con A, F8 gp140 protein, orheat inactivated Ad6. After incubation for 30-36 hours at 37° C., thecells were removed and the wells were thoroughly washed with PBS anddeveloped as per protocol provided by the manufacturer. Results areexpressed as IFN-γ spot-forming cells (SFCs) per 10⁵ PBMCs aftersubtraction of the duplicate wells with medium only (negative control)and are considered positive if greater than twice the background andgreater than 5 SFCs/10⁵ PBMCs.

Antibody ELISAs

HIV-1 envelope binding antibody titers were measured in plasma samplescollected at regular intervals against F8 gp140 or SF162 gp140 asdescribed elsewhere (Malherbe et al., 2014 J Virol 88:12949-12967; andHessell et al., 2016 J Immunol 196:3064-3078).

Neutralization Assay

HIV neutralization was performed using the TZM-bl neutralization assayas described elsewhere (Malherbe et al., 2014 J Virol 88:12949-12967;and Hessell et al., 2016 J Immunol 196:3064-3078). All values werecalculated as compared to virus-only wells.

Antibody Dependent Cellular Cytotoxicity (ADCC)

CEM.NKR.CCR5.CD4+-Luc, target cells were infected with 50 ngSHIV_(SF162P3) and cultured for 4 days as described elsewhere (see,e.g., Alpert et al., 2012 PLoS Pathog 8:e1002890). Two-fold serialdilutions of each sample were added to the infected targets for 20minutes at room temperature. CD16-KHYG-1 effector cells were added at a10:1 effector to target ratio and these were incubated for additional 8hours. The cells were lysed and luciferase activity was measured on theBio-Tek plate reader.

Flow Cytometry

Cells collected from rectal and lymph node biopsies were incubatedovernight with 0.2 μg gp140 or media alone in the presence of GolgiPlug™(BD Biosciences, San Jose, CA, USA) for the last 4 hours. After culture,cells were harvested and incubated on ice for 45 minutes with a panel ofhuman antibodies that cross-react with rhesus macaque samples. Thepanels included the following fluorochrome labeled antibodies: CD8(Qdot655), α4β7 (PE) and CXCR5 (PE), all obtained from the NonhumanPrimate Reagent Resource; CD69 (BV737, clone FN50) and FoxP3 (PECy5,clone: PCH101) obtained from eBioscience, IL-21 (BV421, clone:3A3-N2.1), CD45 (BV786, D058-1283) and CD3 (clone SP34-2,PE-Cy7-labeled) all from BD Bioscience (San Jose, CA); CD4 (PacificBlue, clone OKT4) from ThermoFisher Scientific (Waltham, MA). Dilutionsfor antibodies were determined by following manufacturer'srecommendations. Dead cells were excluded by using live-dead fixabledead cells stain kit obtained from Invitrogen (Carlsbad, CA).Subsequently, the cells were washed twice with PBS containing 2% FBS and2 mM EDTA and then fixed and permeabilized with FoxP3 Fix/Perm Kit(ThermoFisher Scientific, Waltham, MA). The intracellular markers FoxP3and IL-21 were stained in permeabilization buffer. Both compensationcontrols (OneComp eBeads, (ThermoFisher Scientific, Waltham, MA) andfluorescence minus one (FMO) controls were utilized. All the sampleswere collected on an LSR Fortessa X-20 analyzer (BD Biosciences, SanJose, CA) and were analyzed using FlowJo software (FlowJo, LLC, Ashland,Oregon). Approximately 2×10⁵ to 1×10⁶ events were collected per sample.

SHIV_(SF162P3) Rectal Challenge

SHIV_(SF162P3) virus was derived from R157 harvest 3 (3.16.12). Thisstock had a P27 content of 66 ng/ml, RNA content Log ˜9.35, TCID50 inIndian origin rhesus PBMC: 1288/ml, and TCID50 in TZM-bl cells:4.1×10⁴/ml. 1 ml of a 1:300 dilution of the stock was used. This equaled4.3 TCID50 on rhesus PBMCs and 137 TCID50 on TZM-bl cells. This dose wasused for weekly intrarectal (IR) challenge. Plasma samples were analyzedfor SHIV viral RNA copy numbers by Leidos Biomedical Research, Inc.,Frederick National Laboratory. Animals with RNA copies above 10 wereconsidered to be infected and the number of challenges required toinfect that animal were used as events for Kaplan-Meier survivalanalysis. Once infected, the animal was no longer challenged. Plasmaviral loads were monitored periodically by the same method until the endof the study.

SHIV_(SF162P3) Viral Load in Tissues

At the end of study PBMCs and post-mortem tissues were collected. PBMCand gut samples were analyzed for SHIV_(SF162P3) viral RNA by qPCR.Prism 7 Graphical software was used for all statistical analyses.

SC-Ad Expressing HIV-1 gp160

Clade B envelope protein sequences (G4 gp160) were identified before andimmediately preceding a peak in the expansion of antibody neutralizationbreadth (F8 gp160) from HIV patient VC10014. These gp160 sequences wereinserted into SC-Ad6 and SC-Ad657 under the control of the strongcytomegalovirus promoter (FIG. 24A). Ad57 is a species C human Ad thatis nearly identical to Ad6 with variation in its hexon hypervariableregions (HVRs) and in its E3 immunevasion genes (FIG. 24B). Most Adneutralizing antibodies target Ad's hexon HVRs (Pichla-Gollon et al.,2007 J Virol 81:1680-1689; and Sumida et al., 2005 J Immunol174:7179-7185). Given this, Ad6's HVRs were replaced with those fromAd57 to generate a chimeric species C Ad vector termed Ad657. BothSC-Ad6 and SC-Ad657 retain all Ad genes including E1 and lack functionalpIIIA and E3 genes (FIG. 24A). Both SC-Ads can therefore replicate theirgenomes to amplify gp160 expression, but do not generate progeny Adviruses. Both viruses were rescued and produced in 293-IIIA cells andpurified on CsCl gradients. When used to infect A549 cells, both vectorsproduced gp160 as determined by Western blotting.

Different Ad vectors were previously tested in rhesus macaques by thesystemic intramuscular (IM) route and by a variety of mucosal routesincluding oral gavage, oral enteric coated capsules, intranasal (IN),and intravaginal (IVAG). Testing of SC-Ad-G4 by IM, IN, and IVAG routesin small animals revealed that priming by IVAG route generatednegligible antibody responses. In contrast, IN immunization in both miceand hamsters generated strong antibody responses. Given these data andthe potential difficulty in performing IVAG immunizations in humans, theIN route was selected for the mucosal immunization route in thesubsequent macaque studies.

Single Mucosal and Systemic Immunization in Rhesus Macaques

2×10¹⁰ vp of SC-Ad6-G4 Env was used to vaccinate groups of 8 femalerhesus macaques by single IM or IN immunization (FIG. 14 ). This dose isrelatively low, being approximately 7.5-fold lower than recent use ofRC-Ad HIV envelope vaccines delivered by mixed IN and IM immunization. Anegative control vector group was immunized IN with SC-Ad6 expressingEbola glycoprotein (gp). Four weeks later, plasma samples were assayedfor Env binding antibodies against F8 gp140 (FIG. 14 ). This showedsignificantly higher midpoint binding titers in the IM immunized routegroup after single immunization (p<0.01 by ANOVA). SF162 neutralizingantibody (NAb) titers were also elevated at this time point, but did notreach significance by ANOVA for the individual route groups. IM vs. INBoost with SC-Ad6 at Week 4

It was been reported that anti-adenovirus neutralizing antibodies thatare produced by one Ad IM immunization can be avoided by boosting by adifferent route (Xiang et al., 2003 J Virol 77:10780-10789). To testthis route concept to enable the re-use of the same Ad serotype inmacaques, each SC-Ad6-primed group was divided into 2 groups of 4. Thesewere each boosted with SC-Ad6 expressing the alternate F8 Env at week 4by either the IM or the IN route. Plasma samples collected 3 weeks afterthis boost showed elevations in midpoint binding titers in the animalsthat were prime-boosted by the IM-IM, IM-IN, and IN-IM groups. Nodetectable antibodies were observed in the IN-IN group (FIG. 14 ).

SC-Ad657 Boost at Week 13

The animals were then boosted by serotype-switching with SC-Ad657expressing G4 Env at week 13. The same route was used as in the previousboost. Week 15 titers showed that IM primed animals had elevated Envbinding titers near 350, but these levels were not significantlydifferent than controls (FIG. 14 ). In contrast, antibodies in theIN-IM-IM group were significantly higher than both the vector controland the IN-IN-IN group (p<0.01). The IN-IN-IN group again showed no Envantibodies even after 3 immunizations.

Recombinant Trimeric Env Protein Boost at Week 24

Most HIV vaccine studies augment Ad immunizations with protein boosts toamplify antibody responses. For example, in a recent study, RD-Ad26vectors were used twice and boosted three times with adjuvanted gp140protein (Barouch et al., 2015 Science 349(6245):320-4). In an effort todetermine whether this strategy would enhance the SC-Ad vaccines, all ofthe SC-Ad-Env groups were boosted with 50 μg of recombinant F8 trimericgp140 protein mixed with ADJUPLEX™ adjuvant by the IM route. The F8trimeric protein boosted midpoint binding titers by two orders ofmagnitude in all of the groups (FIG. 14 ). This protein immunizationalso boosted the IN-IN-IN to levels comparable to the other groups eventhough Env binding antibodies were not detected after the earlier SC-Adimmunizations.

Binding and Neutralizing Antibodies in Plasma after a Second ProteinBoost

The animals were boosted with protein a second time at week 38. Thisincreased F8 binding plasma antibody titers to nearly 10⁵ by week 40 andall groups became significantly different than controls (FIG. 14 ).Neutralizing antibody (NAb) titers against Tier 1A SF162 virus wereincreased to 100 to 10,000 at week 40 (FIG. 15 ). NAbs against Tier 1Bvirus SS1196 and Tier 2 JRCSF virus increased to 100 in most animalswith the exception of two animals in the IN-IN-IN group whose titerswere at background levels (FIG. 15 ).

ADCC Activity after the Second Protein Boost

Antibody-dependent cellular cytotoxicity (ADCC) activity in week 40plasma was tested against SHIV_(SF162P3) infected cells. ADCC activitywas generally higher in animals that had at least one IN mucosal SC-Adimmunization (FIG. 16 ). All animals that received a mucosalimmunization had significantly higher maximum % ADCC than SC-Ad-Ebolacontrol animals (p<0.05, 0.0001, 0.0001 for IM-IN-IN, IN-IM-IM, andIN-IN-IN, respectively). When compared by 50% ADCC titers, only the INSC-Ad primed groups had significantly higher ADCC activity than controls(p<0.05 and 0.001 by ANOVA for (IN-IM-IM and IN-IN-IN groups).

Antibody Responses in Saliva and Vaginal Washes after a Second ProteinBoost

The data above monitored systemic antibody responses in plasma. Salivaand vaginal wash samples were also collected at week 40 and measured forantibodies in these mucosal sites. When saliva and vaginal washes wereassayed for F8 and SF162 env binding by ELISA, these responses wereobserved in most groups with the exception of the SC-Ad-Ebola controlgroup (FIG. 25 ).

There appeared to be a regional effect on these mucosal antibodies. Inanimals that were immunized with SC-Ad mostly by the IN route (IM-IN-INand IN-IN-IN), binding antibodies were higher in the saliva near thissite of immunization, but lower in the more distant vaginal site (FIG.25 ). When ADCC activity was measured in these mucosal samples, theseresponses were highly variable (FIG. 17 ). Despite this, higher ADCCactivity was observed in the IN-IN-IN group when compared to controlanimals (p<0.05 by ANOVA).

Systemic Cellular Immune Responses after One Protein Boost

Week 38 PBMCs were assayed for T cells against Env and againstadenovirus by ELISPOT on samples collected just prior to a second F8 Envprotein boost. All Env-immunized animals had Env-specific IFN-γsecreting cells in their PBMCs (FIG. 18A). The level of Env-specificIFN-γ SFCs were generally increased in animals that received at leastone mucosal immunization. However, IFN-γ SFCs were only significantlyhigher only in the IN-IN-IN SC-Ad group when they were compared to SC-AdEbola immunized control animals (p<0.05 by ANOVA). Anti-Ad SFCs wererelatively low in all groups when compared to anti-Env SFCs at this timepoint.

Systemic Cellular Immune Responses after a Second Protein Boost

At week 40, PBMCs and inguinal lymph node cells were assayed forEnv-specific IFN-γ SFCs by ELISPOT (FIG. 18B). This protein boostincreased Env-specific SFCs in PBMCs and in lymph nodes to similarlevels in all of the Env-immunized animals.

Mucosal Cellular Trafficking

Flow cytometry on rectal biopsy samples at week 40 showed similarnumbers of α4β7 CD4 and CD8 cells in rectal sites (FIG. 19A). There wasa trend towards increasing numbers in the IN primed groups, but thesedid not reach significance. The numbers of activated CD69+CD4+ cells inrectal tissues were similar between the groups (FIG. 19B). Similarly,FoxP3+CD4+ cells in this mucosal site were not appreciably different(FIG. 19B).

Antigen-specific Tfh Cell Distributions

CXCR5+ IL-21+ CD4+ T follicular helper (Tfh) cells were measured inPBMCs and lymph node samples at week 40 (FIG. 20 ). The animals thatwere immunized by Ad and protein by only the IM route had significantlyhigher peripheral Tfh (pTfh) cells in PBMCs than other groups (FIG. 20). In lymph nodes, Tfh cells were lowest in the control and IM onlygroup. In contrast, approximately one half of the animals that receivedat least one IN mucosal immunization have detectable Tfh in their lymphnodes after the last protein boost (FIG. 20 ).

Rectal Challenge with SHIV_(SF162P3)

The immunized macaques were challenged rectally with HIV isolateSHIV_(SF162P3). Four unimmunized control animals were added to the studyand each group was challenged weekly by rectal inoculation with 1 ml a1:300 dilution of SHIV_(SF162P3) challenge stock provided by NIH. Thischallenge equaled 4.3 TCID50 on rhesus PBMCs and 137 TCID50 on TZM-blcells. After the first challenge, 2 animals in an un-immunized controlgroup and 2 animals in the IM-IM-IM group became infected (FIG. 21 ).One animal in each of the mixed route groups (IM-IN-IN and IN-IM-IM)became infected after one challenge. None of the animals in the IN-IN-INgroup were infected after the first challenge. Viral loads in plasmaindicated that all animals except the Ebola group animal reached highviral loads after 3 challenges (FIG. 22B). Animals in the IN-IN-IN grouphad a delay in reaching these high viral loads.

As challenges continued, animals in all groups became infected with theexception of one animal in the Ebola group that remained uninfectedafter 7 challenges. Trim5α and MHC alleles were examined retrospectively(Table 2). This analysis did not reveal overtly protective genes in theresistant Ebola group animal. Most animals could not be classified withalleles that might keep them moderately protected, but most groups hadat least one animal with a higher likelihood of protection by virtue ofthese alleles. It should be noted that 2/4 animals in the IM-IM-IM andIN-IN-IN groups had Trim5α and MHC alleles that might predict a higherlikelihood of innate protection against SIVsmm and perhapsSHIV_(SF162P3) (Table 2).

TABLE 2 Retrospective Screening for SIV Protective Gene Alleles. Degreeof Vaccine Animal viral Group Number MHC typing TRIM5alpha protectionUnimmunized RHJ663 Not done Cyp A/TFP High RH3-39 Not done Q/TFPModerate RHJ403 Not done CypA/Q Moderate RHJ791 Not done Q/TFP ModerateSC-Ad-Ebov RH13-005 A11, B01, B17 Q/TFP Moderate A08, A11, B01, RH13-007B17 Q/TFP Moderate RH13-043 A08, A11, B17 Q/TFF Moderate RH13-135 A08,A11, B17 Q/TFP Moderate IM-IM-IM RH13-027 A11, B01, B17 TFP/TFP HighRH33-031 A08, A11, B01 Cyp A/Q Moderate RH13-051 A08, A11, B17 Cyp A/QModerate RH13-139 A08, A11, B17 TFP/TFP High IM-IN-IN RH13-039 A11, B01,B17 Cyp A/Q Moderate A08, A11, B01, RH33-045 B17 Q/Q SusceptibleRH13-095 A08, A11, B17 Q/TFP Moderate RH33-159 A08, A11 Cyp ATFP HighIN-IM-IM RH13-013 A11, B17 Cyp A/Q Moderate A08, A11, B01, RH13-067 B17Q/TFP Moderate RH13-091 A11, B01, B17 TFP/TFP High RH13-121 A08, A11,B17 Q/TFP Moderate IN-IN-IN RH13-025 A11, B17 Cyp A/Q Moderate RH13-033A08, A13, B17 Cyp A/TFP High A08, A11, B01, RH13-087 B17 TFP/TFP HighRH13-125 A08, A11, B17 Q/TFP Moderate

When the animals were grouped based on whether they were primed by theIM or IN route with SC-Ad and Kaplan-Meier survival was analyzed,infection of the eight IM primed animals paralleled that of controlanimals (FIG. 22A). In contrast, infection was somewhat delayed in theeight animals that were primed with SC-Ad-Env by the mucosal IN route.

Post-Mortem Viral Loads in Tissues

The challenge study was terminated 9 weeks after first challenge. PBMCsand gut tissues were isolated, RNA was purified, and evaluated for SHIVviral genomes (FIG. 22B). Post-mortem PBMCs had varied levels of SHIVviral RNA with somewhat lower levels in the IN-IN-IN group than in theIM-IM-IM group. Mean viral RNA in the colon was 15-fold lower in theIN-IN-IN group than the IM-IM-IM group (FIG. 23 ). This difference didnot reach significance by ANOVA, but two-tailed T test gave a p value of0.0079.

This example demonstrates that replicating SC-Ad vectors can be used asa robust and safe platform for vaccination against HIV-1 and otherinfectious diseases. SC-Ad is able to amplify antigen and cytokine genesup to 10,000-fold in infected human cells. The immune response isamplified well-above those mediated by RD-Ad vectors that are currentlybeing tested as HIV-1 vaccines in humans. HIV vaccines can betransitioned to vaccine platforms that amplify HIV antigen genes byutilizing SC-Ad vectors, for example, SC-Ad vectors based on recombinantAds having low seroprevelance.

Example 8. In Vivo Cytotoxic T Lymphocyte (CTL) Assay for ImmuneResponses Against Hepatitis C Virus (HCV) Antigen

Mice were immunized with Ad657 expressing the CMV cytomegalovirus (CMV)glycoprotein B (gB) cDNA or HCV antigen 2.4. Syngeneic cells were pulsedwith HCV peptide and labeled with carboxyfluorescein succinimidyl ester(CFSE) prior to injection into the immunized mice. Cognate CTL activityis observed against HCV by loss of labeled cells in the HCV, but not CMVimmunized animals (FIG. 26 ).

Example 9. Conditionally Replicating Ads (CRAds)

Schematic of mutations in Ad6, Ad657 and variants thereof involvingmutations in the E1 protein to convert the virus to aconditionally-replicating Ad (CRAd) is shown in FIG. 39 and FIG. 43 .These include dl1101 and/or the dl1107 that block binding to p300 andpRB, respectively.

FIG. 56 shows the N-terminal amino acid sequences of E1A in a wild-typeAd, as well as Ad variants E1A dl1101, E1A dl1107 and E1A dl1101/1107.

Also shown is the replacement of the Ad E1 promoter with theprostate-specific promoter probasin-E1 DNA sequence of SEQ ID NO:48 togenerate the CRAd, Ad-PB (FIG. 55 ). The probasin promoter is androgendependent, so will work in androgen-sensitive tumors like LNCaP, but notin androgen-resistant tumors like DU145.

A549 cells were infected with the indicated Ad6 or Ad657 variants at theindicated concentrations of virus (vp/cell) and cell viability wasmeasured by crystal violet staining after 5 days (FIG. 40 ).

Killing of non-cancerous cells by replication-defective Ad (RD-Ad), Ad6,CRAd6-dl1101/dl1107 or CRAd6-PB. Modification of Ad6 and Ad657 to beconditionally-replicating Ads (CRAds) is demonstrated (FIG. 44 ).

Killing of cancerous cells by replication-competent Ad5, Ad6, Ad657, andthe indicated CRAds is shown in FIG. 45 . The modification of Ad6 andAd657 to be conditionally-replicating Ads (CRAds) is demonstrated.

The results shown in FIG. 46 demonstrate modification of Ad6 and Ad657to be conditionally-replicating Ads (CRAds) in breast cancer cells.

The results shown in FIG. 47 demonstrate modification of Ad6 and Ad657to be conditionally-replicating Ads (CRAds) in prostate cancer cells andlung cancer cells.

In vivo effects of replication-competent Ad6 or the indicated CRAds ongrowth of DU145 tumors in mice. FIG. 48 demonstrates modification of Ad6and Ad657 to be conditionally-replicating Ads (CRAds) in vivo after asingle intravenous injection in mice bearing human prostate tumors.

In vivo effects of replication-competent Ad6 or the indicated CRAds onsurvival of mice with DU145 tumors. FIG. 49 demonstrates modification ofAd6 and Ad657 to be conditionally-replicating Ads (CRAds) in vivo aftera single intravenous injection in mice bearing human prostate tumors.

FIG. 51 demonstrates that modification of Ad657 with the shorter fiberfrom chimpanzee AdC68 reduces efficacy.

In an embodiment, an Ad 6/57/6 virus has HVRs 1 and 7 from Ad6 and HVRs2-6 from Ad657. FIG. 52 demonstrates Ad 6/57/6 virus killing human lungcancer cells with and without CRAd modifications.

Tumor cell killing by Ad variants involving mutations in the E3 protein.Immune competent Syrian hamsters were engrafted with subcutaneous HaKkidney cancer tumors. When these reached 200 μl volume, they wereinjected a single time by the intravenous route with the indicated Ad6viruses constructed with and without E3 (DE3) and with or without randomNHS-PEGylation. Tumor sizes were measured over time. The data shows thatdeleting all E3 genes makes the oncolytic virus less effective(Ad6-deltaE3-Luc vs Ad6-Luc) (FIG. 58 ).

The Ad fiber protein is a complex of three apparently identical subunitswhich mediates the initial attachment step. The native Ad6 fiber proteincomprises the amino acid sequence set forth in SEQ II) NO:60 and bindsCAR.

In a further aspect of the invention, fiber-modified recombinant Adshaving different fiber proteins which are not native to the parental Adwere generated. Recombinant Ads, including CRAds, comprising capsidproteins from different Ad strains were generated, for example,recombinant Ads comprising a heterologous Ad35 fiber polypeptide orChimpanzee C68 fiber polypeptide, +/−a K7 peptide (FIGS. 62-69 ).

A chimeric Ad, AdF35 fiber chimera, has the amino acid sequence of SEQID NO:61 and is shorter than Ad5 and Ad6 fiber proteins and retargetsvirus to CD46.

A fiber-modified recombinant Ad, comprising K7 Fiber having the sequenceof SEQ ID NO:62, targets virus to heparin sulfate proteoglycans andnegative charges on cells.

A recombinant, chimeric Ad, 6/FC68 Fiber comprising the sequence of SEQID NO:63, is a chimeric Ad having a fiber protein from chimpanzeeadenovirus C68. The fiber protein is shorter than Ad5 or Ad6 fiberproteins and binds CAR.

A recombinant, chimeric Ad, 6/FC68-K7 Fiber comprising the sequence ofSEQ ID NO:64, is a chimeric Ad having a fiber protein from chimpanzeeadenovirus C68. The fiber protein is shorter than Ad5 or Ad6 fiberproteins. The 6/FC68-K7 Fiber binds CAR and is retargeted to heparinsulfate and negative charges.

A recombinant, chimeric Ad, 6/FC68-HI-K7 Fiber comprising the sequenceof SEQ ID NO:65, is a chimeric Ad having a fiber protein from chimpanzeeadenovirus C68. The fiber protein is shorter than Ad5 or Ad6 fiberproteins. The 6/FC68-HI-K7 Fiber binds CAR and is retargeted to heparinsulfate and negative charges.

Example 10. Serotype-Switching of Adenoviruses

FIG. 41 is a schematic showing Ad therapeutic cycles. In an embodimentserotype-switching with different Ads over the course of a treatment isexemplified (FIG. 41A).

Prostate Tumor Targeting after Serotype-Switching of OncolyticAdenoviruses.

Mice bearing DU145 prostate tumors on their flanks were treated by asingle intravenous (IV) injection with Ad657 or CRAd657. These mice weretreated a second time with alternate Ad6 oncolytic virus or Ad6-F35expressing GFPLuciferase and luciferase activity was measured byimaging. Ad6 has Ad6 hexon and fiber that targets CAR. Ad6-F35 has Ad6hexon and the Ad35 fiber that targets CD46. FIG. 42 demonstrates thecapability to serotype-switch oncolytics with viruses targeting a tumorwith lower off-target infection of the liver.

In another example of serotype-switching, mice bearing LNCaP prostatetumors on their flanks were treated by a single intravenous (IV)injection with 3e10 viral particles (vp) of Ad657 or CRAd657. These micewere treated a second time 5 months later with 3e10 vp alternateAd6/57/6 oncolytic virus expressing GFPLuciferase and fiber variants K7(with 7 lysines added), F35 (with the Ad35 fiber), or KKTK-C68(chimpanzee C68 fiber fused after the Ad6 KKTK flexibility domain.KKTK-C68 virus also has an added codon-optimized E4 34K gene to enhanceviral productivity. Luciferase activity was measured by imaging 7 dayslater. All Ad6/57/6's have a hexon with HVR1 and 7 from Ad6 and HVRs 2-6from Ad57. Ad6/57/6 and KKTK-C68 have fibers that targets CAR.Ad6/57/6-F35 has the Ad35 fiber that targets CD46. K7 increases bindingto negative charges on cells including binding heparin sulfateproteoglycans. FIG. 70 demonstrates the capability to serotype-switchoncolytics with viruses targeting a tumor with lower off-targetinfection of the liver.

Serotype-Switching During Vaccination of Non-Human Primates.

In FIGS. 14 through 25 , rhesus macaques were immunized with replicatingsingle-cycle Ad6 expressing HIV envelope and then boosted byserotype-switching with single-cycle Ad657 expressing HIV envelope.Following these immunizations, each animal was boosted with envelopeprotein. Each figure shows the generation of adaptive antibody orcellular immune responses and how the animals repelled rectal challengewith SHIV SF162P3 virus. FIG. 14 documents the value of theserotype-switch where changing to Ad657 generated marked increases inantibody responses.

Example 11. Oncolytic Cancer Vaccines

BALB/c mice were immunized with 10¹⁰ virus particles ofCRAd-657-dl1101/1107-FolR with intact E3 and expressing the human folatereceptor alpha or with PBS by the intramuscular route. Sera wascollected 2 weeks after one immunization and analyzed for anti-FolateReceptor alpha antibodies by ELISA using anti-IgM antibody for detection(all antibodies are IgM at this type of early time point afterimmunization). Data shows the generation of antibodies against the knowncancer antigen folate receptor alpha by this CRAd. p—0.07 by T test(FIG. 53 ).

Example 12. Effects of E3 Immune Evasion Genes on Oncolytic Activity

FIG. 57 shows as schematic of different E3 immune evasion genes in Ads.E3 19K protects infected cells from T cells and NK cells. RID proteinsprotect infected cells from death-inducing ligands (FAS, TRAIL, TNFR,and EGFR). 14.7K inhibits intrinsic activation of apoptosis in infectedcells. Species C Ads also express the 11.6K known as the adenovirusdeath protein (ADP). Over-expression of ADP accelerates cell death, butoverall cell death is equal. Species 49K binds to CD46 on T cells and NKcells leading to down-regulation of these cells and less-efficient cellkilling of cells deficient in class I MHC by NK cells.

FIG. 58 demonstrates that partial deletion of E3 12.5K and full deletionof E3 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ), and 14.7Kgenes reduces oncolytic efficacy in an immunocompetent hamster model ofkidney cancer when these immune evasion genes are not present inoncolytic adenovirus.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A recombinant adenovirus (Ad) comprising nucleic acids which encodeone or more immunostimulatory polypeptides, and wherein the recombinantadenovirus (Ad) capsid hexon polypeptides are from an Ad strain Ad6 andwherein the capsid hexon polypeptides comprise at least two capsid hexonhypervariable region (HVR) polypeptides from Ad strain Ad57.
 2. Therecombinant adenovirus (Ad) of claim 1, wherein the one or moreimmunostimulatory polypeptides is selected from 4-1BBL, humangranulocyte macrophage colony stimulating factor (hGMCSF), CD40L andIL-21.
 3. The recombinant Ad of claim 1, wherein the capsid hexonpolypeptides of the Ad strain Ad6 comprise capsid hexon hypervariableregion (HVR) polypeptides 1-7 from Ad strain Ad57.
 4. The recombinant Adof claim 3, which is Ad657.
 5. The recombinant Ad of claim 1, whereinthe capsid hexon polypeptides of the Ad strain Ad6 comprise capsid hexonhypervariable region (HVR) polypeptides 2-6 from Ad strain Ad57.
 6. Therecombinant Ad of claim 5, which is Ad6/57/6.
 7. The recombinant Ad ofclaim 1 which is a conditionally-replicating Adenovirus (CRAd) which hasbeen modified in an E1A gene encoding an E1A polypeptide, wherein theCRAd exhibits amino acid substitutions in the E1A polypeptide relativeto wild-type E1A polypeptide of an Ad strain.
 8. The recombinant Ad ofclaim 7, wherein the recombinant Adenovirus (Ad) has been modified in anE1A gene to comprise a dl1101 deletion in a nucleic acid encoding an E1polypeptide, modified to comprise a dl1107 deletion in a nucleic acidencoding an E1 polypeptide, or modified to comprise a dl1101 deletionand a dl1107 deletion in a nucleic acid encoding an E1 polypeptide. 9.The recombinant Ad of claim 8, wherein the N-terminal portion of the E1Apolypeptide comprises an amino acid sequence set forth in SEQ ID NO:43,SEQ ID NO:44 or SEQ ID NO:45.
 10. A pharmaceutical compositioncomprising the recombinant Ad of claim 1 and a pharmaceuticallyacceptable carrier, filler, and/or vehicle.
 11. A method for treatingcancer in a mammal, comprising administering to a mammal, therecombinant adenovirus (Ad) of claim
 1. 12. The method of claim 11,wherein the cancer is selected from the group consisting of prostatecancer, ovarian cancer, lung cancer, hepatocellular carcinoma,pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer,lymphoma, myeloma, lymphocytic leukemia, and myelogenous leukemia. 13.The method of claim 11, wherein the administering comprises systemicadministration.
 14. The method of claim 13, wherein the systemicadministration comprises intramuscular, intranasal, or intravenousadministration.
 15. The method of claim 11, wherein the administeringcomprises local administration.
 16. The method of claim 15, wherein thelocal administration comprises intratumoral injection.
 17. The method ofclaim 11, further comprising administering one or more additional agentsused to treat cancer.
 18. The method of claim 17, wherein the one ormore additional agents used to treat cancer is selected from the groupconsisting of chemotherapy, hormone therapy, targeted therapy, andcytotoxic therapy.